•The chitosan/rGO/MnO2 based working electrode is able to provide an increase in sensor work activity.•Adding MnO2 in chitosan film could be potentially for sensor material to detect at the lowest concentration.•Sensing properties increases with increasing variation of MnO2. Cholesterol detection needs to be carried out to detect early symptoms of dangerous diseases such as heart disease. The process of detecting cholesterol in the human blood requires a sensitive detection tool in order to produce accurate values. This paper reports the performance of chitosan thin films modified with reduced graphene oxide (rGO) and manganese dioxide (MnO2) as the sensitive layer of working electrodes in detecting cholesterol. The electrode substrate is a copper-screen printed electrode (copper-SPE) which was patterned into 5 mm of sensitive area. The chitosan and chitosan-reduced graphene oxide (rGO) matrices were deposited on the top of working electrodes. The characterization of chitosan, chitosan/rGO, and chitosan/rGO/MnO2 thin films was performed using AFM, FE-SEM, and FTIR. The electrochemical sensing properties of modified working electrode were observed using an electrochemical method of cyclic voltammetry (CV). The novelty of this research is the utilization of chitosan/rGO/MnO2 thin film-based working electrode for detecting cholesterol. The testing results indicated that the sensitivity of the modified working electrodes increased when working electrodes based on chitosan/rGO/MnO2 thin film were used for cholesterol detection compared to the working electrodes based on the chitosan thin film. It was proven from the sensitivity of working electrodes based on chitosan/rGO/MnO2 thin film which gave the lowest value of 3.21 × 10−5 µA/µM as well as the ability of working electrodes based on chitosan/rGO/MnO2 thin film which worked at limit of detection (LoD) 0.26 µM/L and limit of quantification (LoQ) 0.87 µM/L. Based on the sensitivity, LoD, and LoQ values, the chitosan/rGO/MnO2 modified electrodes can be used as working electrodes to measure cholesterol concentration in blood.