Proteolytic bacteria that are potential to produce protease enzymes are widely used in food processing which can be isolated from various parts of fish and from various processes, which can be isolated from fish entrails, fresh fish and processed fish such as anchovy, and fish canning process waste. The study of proteolytic bacteria in anchovy has great biotechnology potential to be investigated because bacteria that can grow in anchovy are bacteria that can live in extreme environments. Given the great potential of the Bacillus pumilus bacteria, research can be carried out on enzyme activity from bacteria that grow in the isolation of anchovy. The purpose of this study was to obtain Bacillus pumilus protease enzyme activity from anchovy isolates which included temperature. The study used the experimental method, which is called Complete Random Design (CRD) with different temperature treatments including Bacillus pumilus 20 o C, 30 o C, 40 o C, and 50 o C. Based on the results of the study, it was found that the crude extract of the protease enzyme anchovy isolate in Bacillus pumilus was 50 o C at 1.2589 u/mL. The results of the analysis show that the same temperature gives an influence between Bacillus pumilus on enzyme activity carried out to determine the optimum condition of the enzyme in degrading the substrate.