Background <p>Microbial cholesterol oxidase (ChoX) has wide clinical and industrial applications; therefore, many efforts are being made to identify promising sources. This study aimed to isolate a novel ChoX-producing bacterial strain from whey samples.</p> Results <p>The most efficient strain was selected based on extracellular ChoX-producing ability and characterized as <i>Escherichia fergusonii</i> (<i>E. fergusonii</i>) through molecular and biochemical analysis. The maximum production of ChoX was obtained at the optimum condition of 48 h of incubation under shaking conditions (130 rpm) at 35 °C in a basal medium adjusted to pH 6.5, including 1.4 g/L cholesterol as a sole carbon. The crude product was purified by ammonium sulfate precipitation and followed by ion exchange chromatography utilizing Q-Sepharose, resulting in 5.35-fold and 13.86-fold purification, respectively, with a final specific activity of 15.8 U/mg. Additionally, molecular weight was determined by SDS-PAGE to be 49.0 kDa. The optimum conditions required for the higher cholesterol decomposition ability of purified ChoX were suggested to be 30 °C and pH 7.5 in the presence of MgSo₄ with a <i>K</i>_<i>m</i> value of 0.71 mM. However, other case studies of metal ions showed an unfavorable effect on enzymatic performance. The enzyme retained almost 72.0% of its initial activity after 80 days of storage at 4 °C. Furthermore, the ChoX enzyme revealed acceptable stability at a pH value of 6.5 to 8.5, maintaining its initial activity of more than 50.0%. Finally, an artificial neural network (ANN) was designed to predict the most effective factor in the fermentation process for enzyme production and the purified ChoX activity.</p> Conclusions <p>Considering the properties of the extracted enzyme from <i>E. fergusonii</i>, it would be regarded as a potential ChoX source for commercial applications.</p>