In some cases, it could be a new group or organization created specifically for the fanfic. This group might be made up of rogue ninja who are jealous of Naruto's newfound ability or who have their own motives related to bloodlines. They could be trying to either steal Naruto's ability or stop him from using it to protect their own interests in the world of bloodlines and ninja power.
It could be Orochimaru. Since he's always interested in kekkei genkai and bloodlines, he might oppose Naruto when he discovers Naruto's new ability to copy bloodlines. Orochimaru would see Naruto as a threat to his own plans of collecting and using powerful bloodlines.
Well, it could be that in this fanfic, Naruto has developed a new jutsu that allows him to analyze and then replicate the DNA - like aspects of bloodlines. He might be using his vast chakra reserves to fuel this ability. For example, if he encounters a bloodline with a special element - like the Ice Release of the Yuki clan - he could break down the chakra signature and recreate it within himself.
Maybe he uses some kind of special jutsu. It could be a new form of his shadow clone jutsu where the clones are able to analyze and then copy the bloodline traits of the opponents they encounter.
A jealous classmate could be a main antagonist. This person might be envious of Naruto's popularity and his ability to make friends easily. They could try to spread rumors or sabotage Naruto's projects.
The Akatsuki could still be a major antagonist. Even if Naruto is emperor, they might oppose his rule as it goes against their own goals of chaos and obtaining the tailed beasts.
One possible antagonist could be a jealous noble from Naruto's own kingdom who feels that Naruto doesn't deserve the prince title. This person might try to undermine Naruto at every turn, spreading false rumors about him or trying to sabotage his diplomatic efforts.
Another possible antagonist could be a fellow inmate who has a personal vendetta against Naruto. Maybe this inmate was once on the wrong side of Naruto's actions outside the prison and now sees this as an opportunity for revenge. He could try to undermine Naruto at every turn, from spreading false rumors about him to physically attacking him when the guards aren't looking.
Another option could be Orochimaru. He has his own motives and is always looking for power. He could be the head of a powerful gang in the criminal underworld of the Naruto world. He would constantly be trying to capture Naruto or his friends to use them for his experiments or to gain more power over the ninja world.
The Akatsuki could still be the main antagonists. They might be interested in Naruto's tails for their own dark purposes, like using their power to create a super - weapon or gain control over the ninja world. Another option could be a secret group of rogue ninjas who have a vendetta against the Uzumaki clan and see Naruto's tails as a symbol of their power that they want to destroy.
One potential antagonist could be a new and mysterious organization that is similar to Akatsuki but with different motives. They might be interested in using Daisuke to open a portal to a different dimension or something like that. They would be constantly trying to kidnap Daisuke and force him to cooperate. Also, Orochimaru or his remaining followers could be antagonists. Even though Orochimaru has changed somewhat in the series, there could still be some of his followers who are more radical and want to use Daisuke for their dark experiments, believing that he has some special chakra or ability that could help them achieve their twisted goals.
There could be a new, mysterious force that is neither from the Naruto world nor fully part of the Olympian world. This force wants to disrupt the balance between the two worlds and sees Naruto as the key to achieving its goals. It could manipulate both sides and create chaos.