A mixture of commercial cholesterol esterase, cholesterol oxidase and peroxidase was co-immobilized covalently on the surface of a polyvinyl chloride (PVC) sheet (plastic strip) after its chemical modification. The strip retained 75.23% of its initial activity of enzymes with a conjugation yield of 0.37 mg protein cm⁻². The strip bound enzymes showed optimum activity at pH 7.2, when incubated at 45 °C for 5 min. The strip was employed for determination of total cholesterol in serum. Minimum detection limit of the method was 0.01 mM in serum. Analytical recovery of added cholesterol in serum (100 mg dL⁻¹ and 200 mg dL⁻¹) was 92.50% and 93.33% respectively. Within and between assays coefficients of variation (CVs) were 1.85% and 2.04% respectively. A good correlation (r = 0.998) was obtained between serum cholesterol values by standard enzymic colorimetric method and the present method. The strip was reused 200-times over a period of 4 months, when stored at 4 °C. The method has the advantage that it provides the reuse of enzymes with enormous ease. Overall results also strengthen our view of using a chemically modified plastic strip as a solid support for covalent immobilization of enzymes.