•The E. coli was covalently immobilized on nanoparticles with a “click” reaction.•Azides were metabolically integrated onto cell surface for directed immobilization.•The immobilization improved the cell’s activity, stability, and reusability. A method for specific immobilization of whole-cell with covalent bonds was developed through a click reaction between alkyne and azide groups. In this approach, magnetic nanoparticle Fe3O4@SiO2-NH2-alkyne was synthesized with Fe3O4 core preparation, SiO2 coating, and alkyne functionalization on the surface. The azides were successfully integrated onto the cell surface of the recombinant E. coli harboring glycerol dehydrogenase, which was employed as the model cell. The highest immobilization yield of 83% and activity recovery of 94% were obtained under the conditions of 0.67 mg mg−1 cell-support ratio, pH 6.0, temperature 45 °C, and 20 mM Cu2+ concentration. The immobilized cell showed good reusability, which remained over 50% of initial activity after 10 cycles of utilization. Its activity was 9.7-fold higher than that of the free cell at the condition of pH 8.0 and each optimal temperature. Furthermore, the immobilized cell showed significantly higher activity, operational stability, and reusability.