Kaempferol (KA) is a flavonoid with a range of biological properties, including antitumor, antioxidant, antiviral and anti-inflammatory, and its extensive applications in biomedicine, food safety, and related fields underscore the importance of quantitative analysis for determining its concentration. In this study, an electrochemical sensor based on multi-walled carbon nanotubes (MWCNTs), PCN and chitosan (CS) was developed for the determination of KA. MWCNTs exhibit hydrophobicity and conductivity, and they are better dispersed by DMF and crosslinked with PCN, which further improves the electrode's response, selectivity and sensitivity to KA due to the peroxide-like properties of PCN. The film formed by CS on the electrode surface serves to protect the nanocomposite from detaching during the operation. The linear range of this sensor is 0.01-0.4 and 0.6-9 μM, with a detection limit of 4.16 nM. This method can be used to detect the content of KA in plasma, which shows that the electrochemical sensor has strong practical application capabilities, as well as other advantages, such as high stability, strong anti-interference ability, and low detection limit. Moreover, the ultraviolet-visible spectrophotometer (UV) demonstrates that the catalytic rate of PCN for KA is significantly faster than that for naringenin and puerarin. Therefore, the construction of CS/MWCNT-PCN/GCE electrochemical sensors has potential application value for clinical dosage control and monitoring of the drug metabolism of KA.