Peptones are produced from products with high protein content. Recently, peptone produced from fishery by-products were sought as a source of bacteria substrate. In this study, the enzymatic hydrolysis of peptone production of Yellowstrip scad fish at the end post-rigour phase was optimized. Peptones produced were physicochemically characterised and tested for microbial growth application. Proximate composition, peptone characterisation and amino acid analysis were conducted following AOAC methods. The pH value of the peptones was measured using a pH-meter and the microbial growth was monitored via measuring the optical density (OD) of the cultures. The outcome of the research showed that the best enzymatic hydrolysis was 0.20% (w/v) papain carried out for 6 hours. The results showed that the Yellowstripe scad fish peptones (YSFP) had a high protein level (77.46%) and contained essential amino acids such as histidine (4.12%), isoleucine (0.96%), leucine (2.42%), lysine (2.25%), methionine (3.07%), phenylalanine (1.68%), threonine (1.06%), valine (2.61%), and arginine (2.85%). The end post-rigour phase YSFP has a solubility of 97.08%; total nitrogen of 12.39%; α-amino nitrogen of 1.03 g/100 g; α-amino nitrogen per total nitrogen of 8.31 and salinity of 4.19%. Bacillus spp., Staphylococcus aureus and Escherichia coli was observed to have better growth performances on the YSFP than bacto peptone.