A prototype chronoamperometric biosensor for the determination of total cholesterol was developed that consists of a homemade potentiostat and disposable strips immobilized with Fe(3)O(4), cholesterol oxidase (ChOx), and cholesterol esterase (ChE). The principle of sensing cholesterol is based on the detection of reduction signal of hydrogen peroxide generated in two enzymatic reactions. The co-immobilization of ChE and ChOx allows the sensor to detect both concentrations of esterified and free cholesterol. The effects of biosensor on catalyst, enzymes, applied potential, and buffer pH was investigated, and the operation conditions were optimized. The detection of cholesterol can be accomplished in one step, a 10 microL of sample was dropped onto the area of sensing strip and the reduction signal was obtained at an applied potential of -200 mV (vs. Ag/Ag(+)). The pre-reaction time was set at 15s before applying potential on the strip and the sampling time was 5s. The sensing device displays a linear response over the range of 100-400mg/dL (R(2)=0.999) for cholesteryl oleate. The coefficient variation was determined as 5.06% (N=20) for 100mg/dL cholesteryl oleate and the detection limit is 19.4 mg/dL (S/N=3). The probable interferences in bio-matrix were selected to test the selectivity and no significant response was observed in the biosensor.