A facile one-step electrochemical synthesis of a platinum/reduced graphene oxide/poly(3-aminobenzoic acid) (Pt/rGO/P3ABA) nanocomposite film on a screen-printed carbon electrode (SPCE) and its application in the development of sensitive amperometric biosensors was successfully demonstrated herein. The electropolymerization of P3ABA together with co-electrodeposition of rGO and Pt was conducted by cyclic voltammetry, as was the GO reduction to rGO. A Pt/rGO/P3ABA-modified SPCE exhibited excellent electrocatalytic oxidation towards hydrogen peroxide (HO) and can be employed as an electrochemical platform for the immobilization of glucose oxidase (GOx) and cholesterol oxidase (ChOx) to fabricate glucose and cholesterol biosensors, respectively. Under the optimized conditions at a working potential of +0.50 V, the proposed biosensors revealed excellent linear responses to glucose and cholesterol in the concentration ranges of 0.25-6.00 mM and 0.25-4.00 mM, respectively, with high sensitivities of 22.01 and 15.94 μA mM cm and low detection limits (LODs) of 44.3 and 40.5 μM. Additionally, the Michaelis-Menten constant (K) of GOx was 3.54 mM, while the K of ChOx was 3.82 mM. Both biosensors displayed a good anti-interference ability and clearly exhibited acceptable recoveries for the detection of glucose and cholesterol in a human serum sample (98.2-104.1%).