A molecularly imprinted electrochemical sensor based on MXene fibers was proposed in this work. Firstly, the wet spinning technique prepared MXene fibers with a large aspect ratio, which can make the sheet-like MXene uniformly arranged, avoiding the agglomeration of MXene and improving the electrical conductivity. Afterwards, molecularly imprinted polymers (MIPs) with specific recognition sites were synthesized on the surface of MXene fibers using the electro-polymerization method. The electrochemical sensor utilized the advantages of MXene fibers and molecular imprinting techniques to gain superior selectivity and sensitivity of hydrocortisone (HC). Electrochemical tests with different concentrations of HC (0.5 nM-10.0 μM) under optimal measurement conditions exhibited excellent linearity and a limit of detection (LOD) of 0.17 nM. Furthermore, the electrochemical sensor displayed excellent selectivity, interference resistance, reproducibility, stability and outstanding application performance in serum. This work has promising applications in trace analysis in real sample.