© 2021, The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.In this work, the separations and preconcentrations of Zn(II) and Hg(II) ions on Bacillus lichenifoemis loaded onto Amberlite XAD-4 resin by solid-phase extraction has been performed. The biosorbent was characterized by using FT-IR, SEM, and EDX. pH, sample flow rate, eluent type and concentration, amount of B. licheniformis and XAD-4 resin, sample volume, and possible interfering ions effect were investigated in details as experimental variables in the SPE procedure. Limit of detection values for Zn(II) and Hg(II) were detected as 0.03 and 0.06 ng mL−1, respectively. 0.2–15 ng mL−1 linear range values were achieved for Zn(II) and Hg(II), respectively. Relative standard deviation values were found to be lower than 5%. For validation of the procedure, the certified standard reference materials (CWW-TM-D, EU-L-2, NCS ZC73O14, NCS ZC73350) were analyzed. The concentrations of Zn(II) and Hg(II) in water and food samples were measured by ICP-OES. Consequently, it can be inferred that the immobilized B. licheniformis microcolumn has ideal selectivity for Zn(II) and Hg(II) biosorption. Graphical abstract: [Figure not available: see fulltext.].