Background: The ratio of fPSA/tPSA in the “grey zone” of tPSA with the concentration range between 4 ng/ml and 10 ng/ml is significant for diagnosis of prostate cancer, and highly efficiency quantification of the ratio of fPSA/tPSA remain elusive mainly because of their extremely low concentration in patients’ peripheral blood with high biosample complexity.Methods: We presented an interdigitated spiral-based MXene-assisted organic electrochemical transistors (isMOECTs) biosensor for highly sensitive determination of fPSA/tPSA. The combination of MXene and the interdigitated multiple spiral architecture synergistically assisted the amplification of amperometric signal of biosensor with dual functionalizations of anti-tPSA and anti-fPSA.Results: The ultrasensitivity of the biosensor was enhanced by tunable multiple spiral architecture and MXene nanomaterials; and the sensor demonstrated enhanced detection limit of tPSA and fPSA down to 0.01 pg/ml, as well as satisfactory selectivity, reproducibility and reliability, which was comparable to the best previous literatures; Moreover, the isMOECTs displayed area under the curve (AUC) value of 0.8138, confirming the potential applications of isMOECTs in clinics.Conclusions: The merits of isMOECTs biosensor demonstrated the reliability of MXene-assisted organic electrochemical transistor biosensor with multiple interdigitated spiral for ultrasensitive quantification of fPSA/tPSA, suggesting potential current and future point-of-care testing applications.