Proteases have enormous potential in various industries, from food to pharmaceutical. Protease activity is strongly influenced by the substrate content during fermentation, especially amino acids. This study was conducted to determine the effect of skimmed milk and casein substrates on the protease activity produced by the endophytic fungus, Fusarium sp. strain JE-DP4a isolated from Carica papaya L. leaves. Selective isolation of protease-producing fungi was carried out qualitatively using skim milk agar (SMA) medium. Endophytic fungus that produced the highest protein hydrolysis index (PHI) was used for the protease fermentation assay to measure a protease activity. The fermentation was carried out on Czapek-Dox + skimmed milk and Czapek-dox + casein media at 37 °C for seven days. A total of 19 protease-producing fungal endophytes were isolated from C. papaya leaves. Among them, Fusarium sp. strain JE-DP4a exhibited the highest PHI value at 1.52. The highest protease activity was produced on Czapek-dox + casein medium with a protease activity of 46.44 U/mL, while the activity of protease enzyme in Czapek-dox + skim milk medium was 15.28 U/mL.