In order to improve the electrochemical sensing performance toward heavy metal ions, this work presents a novel L-cysteine functionalized mesoporous MnFe2O4 hybrid nanospheres (MnFe2O4@Cys) applied as electrochemical sensor for determination of Pb(II), Hg(II), Cu(II) and Cd(II) based on square wave anodic stripping voltammetry (SWASV) technique. Combined the excellent adsorption capacity toward heavy metal ions of mesoporous MnFe2O4 nanocluster with Lewis acid-base interaction supported by L-cysteine, the MnFe2O4@Cys modified glass carbon electrode (GCE) successfully realized individual and simultaneous detection toward four target ions. In particular, the L-cysteine modified Mn and Fe bimetal oxides nanoparticles displayed good selectivity toward Pb(II) with high sensitivity of 57.0 μA/μM and 35.3 μA/μM under the individual and simultaneous determination conditions, respectively. Meanwhile, this fabricated sensor showed satisfying anti-interference, reproducibility, stability, repeatability and applicability for detecting heavy metal ions. It provides a promising platform for developing new and high performance electrochemical sensors applied in individual and simultaneous determination of various of heavy metal ions.