Taking a composite of a nanomaterial and a signal molecule as a substrate material can provide a label-free electrochemical platform. Besides, the nanomaterial with a high catalytic activity towards the signal molecule can improve the sensitivity of the platform. Herein, a thionine functionalized Fe-N-C nanocomposite was employed as the substrate. Firstly, the electrocatalytic activity of Fe-N-C towards the electroreduction of thionine was explored. Then, an immobilization-free and label-free electrochemical platform for the determination of microRNA-21 based on Fe-N-C-thionine/Fe3O4@AuNPs was constructed. A magnetic glassy carbon electrode (MGCE) was used to keep the magnetic Fe-N-C-thionine/Fe3O4@AuNPs modified onto the surface of the MGCE. Fe-N-C and Fe3O4 nanoparticles can co-catalyze the electroreduction of thionine and a strong electrochemical reduction signal of thionine could be realized in the differential pulse voltammetry (DPV) test. Also, a catalytic hairpin assembly (CHA) reaction was utilized to enhance the sensitivity of the developed electrochemical biosensor. Besides, the developed biosensor shows excellent specificity and reproducibility in the test of human serum samples, indicating its wide application prospects in the early-stage diagnosis of tumors.