Proteolytic bacteria are most important for industries such as food and fermentation. Thus isolating and manipulating pure culture from various sources has importance for various biotechnology industries. In the present investigation a total of 26 bacteria were isolated from the dairy plant effluent. All isolates were screened for proteolytic activity using alkaline skim milk agar plate, among 26 bacterial isolates only 7 isolates showed alkaline proteolytic activity, one isolate was selected for further study. Characteristic feature and 16S rDNA analysis of the strain indicates that the organism was Bacillus subtilis. The culture and medium components was optimized further. The best enzyme activity was observed at pH 9 and temperature 45 °C, 5% MgSO4 concentration, glucose as carbon source and beef extract as nitrogen source. Also maximum enzyme activity was observed when using wheat bran as the substrate. It is well proved that the dairy effluent can be used as the isolation source for alkaline protease production which in further having many industrial applications.