Fish is beneficial for health due to its content of EPA, DHA, linoleic acid, iodine, protein, selenium, omega-3, vitamin A, vitamin D, and vitamin B12. Fish has the potential to be developed into fish oil. According to WHO, the consumption of fish oil can prevent the risk of mortality and improve health functions. However, fish consumption in toddlers is still low. A survey conducted on children in Indonesia showed that 45.1% experienced Vitamin D deficiency. Vitamin D is known to play a role in the immune system as an immunomodulator and enhances innate immunity. This research aims to compare the vitamin D content of fish oil extracts from several types of freshwater fish and determine their fatty acid content. The researched fish will produce different fish oil and vitamin D content, necessitating research to assess the vitamin D content. Furthermore, fish oil with high vitamin D content can be considered a competent candidate for health supplements. Three types of fish, namely snakehead fish, eel, and catfish, were chosen as research samples. The research was conducted at the Integrated Laboratory of Poltekkes Medan and PPKS. This experimental research used a Randomized Block Design (RBD) with three replications. The data were then statistically analyzed using Anova with a significance level of 5%. The research results indicate that the highest vitamin D content is found in eel with alcohol solvent (12.2974 mg/l). The vitamin D content in snakehead fish with alcohol solvent is 7.8832 mg/l, while in catfish with alcohol solvent, it is 3.0069 mg/l. For fish oil with N-Hexane solvent, the vitamin D content in snakehead fish is 1.4096 mg/l, eel is 2.2489 mg/l, and catfish is 4.5706 mg/l.