Objective The fundamental structure of articular cartilage comprises three layers: the upper layer (UL, approximately 20%), the middle layer (ML, approximately 50%), and the lower layer (LL, approximately 30%). Owing to their distinct compositions and structures, the mechano-electric characteristics of these layers differ, this study delves into the analysis of the inverse mechano-electric effect of the layered structure of articular cartilage and its influencing factors. Method The cartilage samples are classified according to their physiological thickness (approximately 0.4 mm for the upper layer, 1 mm for the middle layer, and 0.6 mm for the lower layer). Through a non-contact external electric field testing method, this study aims to analyze how different influencing factors affect the inverse mechano-electric effect of articular cartilage. Results In conditions where the electric field spacing decreases, water content increases, and in vitro time decreases, normal layered cartilage demonstrates an increasing trend by about 18 μm、10 μm、15 μm in displacement generated in a non-contact electric field. In simulated arthritis defect scenarios, as the defect depth and radius increase, the overall deviation deflection of the articular cartilage gradually decreases by about 7μm. Conclusion The three layers of cartilage differed in their inverse mechano-electricity effect, showing the greatest deflection mid-layer at 90% water content, 7 mm electric field spacing, and after 12 h of ex vivo.