The determination of coagulation parameters is of crucial significance in the guided treatment of anticoagulant drug users and patients with liver diseases. Considering the challenges associated with commercial coagulation detection instruments such as high cost and complex operation, a MWCNT-enhanced screen-printed electrochemical sensor was developed for coagulation measurement. Initially, the measurement of thrombin substrate cleaved by thrombin was used to evaluate the feasibility of prothrombin time (PT) detection by chronoamperometry. Then the plasma PT parameters were measured and the results were validated using the SYSMEX CS 5100 optical coagulator. The response intensity of a MWCNT electrochemical sensor in thrombin validation experiments increase by (36±1)% compared with a general electrochemical sensor and the peak time coefficient of variation and peak current coefficients of variation are 2.99% and 3.27% respectively. The testing of PT values of different blood samples clearly shows discrimination. Three groups of blood samples are selected for repeated PT parameter measurements and the coefficients of variation are 2.26%, 322%, and 2.96% respectively. The linear fitting decision coefficient R2 is 0.986 for clinical results. The MWCNT screen-printed electrochemical sensor for PT testing has good repeatability and consistency, is easy to mass produce, reduces the cost of coagulation measurement, and is suitable for measurement in many circumstances. As such, this sensor has great potential in the field of point-of-care testing.