A single-use, reagentless, screen-printed biosensor strip for the measurement of free cholesterol was fabricated and its performance characteristics were evaluated by cyclic voltammetry and under steady-state conditions. Cholesterol oxidase was adsorbed on the surface of an H2O2-sensing cobalt phthalocyanine screen-printed carbon electrode and retained at the sensory interface with a solvent-cast cellulose acetate membrane. The device, operated in the amperometric mode, provides an inexpensive, reliable and rapid means of quantifying this clinically significant sterol. Its analytical utility is further augmented by its ease of fabrication and favourable storage properties. The biosensor exhibits a wide functional range (6 × 10–5–5.0 × 10–3 mol dm–3) and the sensitivities of the devices (slopes of the calibration graphs) were used to derive optimum solution conditions. Several analytical parameters were also investigated to optimize and enhance the response time and sensitivity of the sensor and these are also discussed.