As a vital therapeutic agent for Parkinson’s disease, dosage control of levodopa has always been a major obstacle in ensuring efficacy and curbing side effects. Simple and fast electrochemical detection methods are the main force in this field. Here, we presented a differential dual-strip method based on the coulometry for high accuracy determination of levodopa. The difference between the two strip signals with or without the tyrosinase extracted the levodopa signal from the samples. The Prussian Blue modified carbon screen-printed electrode was used to convert and amplify the electrochemical signal upon the presence of levodopa. The system exhibited a linear behavior in the 0-10 μM concentration range and a detection limit of 0.25 μM. Furthermore, it was proved to be stable in effectively distinguishing levodopa from complex samples through anti-interference experiments and serum tests. We demonstrated the superiority of dual-strip differential coulometry for the determination of levodopa towards Parkinson’s disease clinical management.Copyright © 2020 Elsevier B.V. All rights reserved.