Cholesterol oxidase (COX) is a key enzyme in diagnostic kits of cardiovascular diseases via oxidation of cholesterol producing smart enantiomerically compounds; however, the enzyme catalytic stability is the challenge. So, the objective of this study was to purify COX from novel endophytic bacterial isolates of medicinal plants that could have unique catalytic efficiency for the desired applications. Among the recovered forty bacterial isolates, Burkholderia gladioli EFBL PQ721377, an endophyte of Eruca sativa, had the highest COX productivity (14.7 μmol/mg/min). The COX productivity of B. gladioli has been maximized by with the response surface methodology, giving the highest productivity 30.9 μmol/mg/min, by ~ 2.0-fold increment compared to control. The enzyme was purified to its molecular homogeneity with subunit structure 40 kDa. The enzyme was entrapped in Ca-alginate with immobilization yield 87.5%, and the efficiency and homogeneity in Ca-alginate beads were assessed by FTIR and SEM–EDX analyses. The free and Ca-alginate-COX conjugates have the same maximum reaction temperature at 37–40 °C, reaction pH at 7.5 and pH stability at 6.5–8.0. The thermal stability of Ca-alginate-COX was increased by ~ 7.0 folds compared to the free one, ensuring the protective role of alginate beads on enzyme tertiary structure. Ca-alginate-COX had a higher potency of oxidation of human serum cholesterol, than the free one, confirming the feasibility of the product release, and allosteric activation of the enzyme, with a reliable operative stability till the fifth cycle, for production of cholest-4-en-3-one, as the precursor of various drugs.