Detection of multiple analytes in biofluids is of significance for early disease diagnosis, effective treatment monitoring, and accurate prognostic assessment. Electrochemical sensors have emerged as a promising tool for the multiplexed detection of biofluids due to their low cost, high sensitivity, and rapid response. Two-dimensional transition metal carbon/nitride MXene, which has the advantages of a large specific surface area, good electrical conductivity, and abundant surface functional groups, has received increasing attention in the electrochemical sensing field. This paper systematically reviews the advances of MXene-based electrochemical sensors for multiplexed detection in biofluids, emphasizing the design of MXene-based electrode materials as well as the strategies for distinguishing multiple signals during simultaneous electrochemical analysis. In addition, this paper critically analyzes the existing challenges of MXene-based electrochemical sensors for multiplexed detection of biofluids and proposes future development directions for this field. The ultimate goal is to improve biofluid multiplexed detection technology for clinical medical applications.