Sardine, Sardinops melanosticta, has been caught more than fifty thousand metric tons every year in adjacent sea of Korea, but most of them used for uneatable fish meal because of their rapid spoilage. Usually it is known that the main reason of putrefaction of foods is caused by the maicro-organisms included in them. Therefore, this experiment was carried out to identify the micro-organisms isolated from the intestine of fresh sardine and characterize their proteolytic enzymes produced from them. Aerobic cell count ranged from $1.7{ imes}10^4;to;3.6{ imes}10^5/g$, while anaerobic cell count, from $2.9{ imes}10^4;to;5.5{ imes}10^5/g$. Most of the isolated strains were psychrotrophic mesophiles. Among the two hundred and eighty strains isolated from the fresh samples, fifty-six strains ($20.0\%$) were proteolytics, one hundred and seventy-five strains ($62.5\%$) were lipolytics and tenty-nine strains ($10.5\%$) had the ability to produce hydrogn sulfide. The most predominantly isolated microbial groups from the fresh sardine were Moraxella ($31.4\%$) and Pseudomonas sup. ($28.6\%$). Flavobacterium-Cytophaga, Vibrio, Acinetobacter, Micrococcus spp. and Enterobacteriaceae appeared from $7.9\%;to;5\%$ out of total tested strains. The average bacterial count in the spoiled samples (stored at about $18^{circ}C$ for 48 hours) was increased to the level of $2.9{ imes}10^8/g$ for aerobes, $1.5{ imes}10^8/g$ for anaerobes, then one hundred and ten strains, corresponding to $52\%$, out of two hundred and thirteen strains submitted to the test were proteolytics. The strongest proteolytic bacterium among the two hundred and eighty strains was identified as Pseudomonas 101 which grew best at $25^{circ}C$. The optimum condition for the activity of the proteolytic enzyme produced by Pseudomonas 101 appeared $35^{circ}C$ and pH 9.0, but the activity was relatively unchanged between 5.0 and 11.0 of pH and between $30^{circ}C;and;50^{circ}C$ of temperature.