The giant gourami (Osphronemus goramy), rice eel (Monopterus albus), and mackerel tuna (Euthynnus affinis) are among the sources of protein and albumin that have been used to lower the blood glucose level of people with Diabetes Mellitus (DM). This study aims to determine the protein levels and albumin content of O. goramy, M. albus, and E. affinis and the inhibition activity of albumin against the α-glucosidase enzyme. This study employed an experimental method with accidental sampling. The protein content of fresh fish fillets was tested using the Kjeldahl method and extracted using a centrifuge to obtain albumin. Albumin levels were determined using a visible spectrophotometer. The inhibitory activity of albumin against α-glucosidase enzyme was tested using an ELISA reader. The results showed a significant difference between freshwater and seawater fish proteins, namely giant gourami of 13.91%, rice eel of 14.41%, and mackerel tuna of 30.55%. The highest albumin content was obtained from mackerel tuna (4.75 ± 0.04 g/100 mL), followed by giant gourami (3.61 ± 0.26 g/100 mL), and rice eel (2.38 ± 0.26 g/100 mL). The albumin showed no significant activity against the α-glucosidase enzyme.