A whole-cell-based amperometric biosensor was fabricated using Escherichia coli cells immobilized on a glassy carbon electrode to detect mercury, cadmium, and zinc ions. E. coli cells were immobilized by the cross-linking method using bovine serum albumin (BSA) in glutaraldehyde (GA) vapor. The principle of the microbial electrode sensor is the inhibition of alkaline phosphatase (AP) enzyme activity by heavy metal ions. Therefore, by monitoring the oxidation current of the product generated by AP in a metal ion solution, its concentration was determined. The Michaelis–Menten constant (Km) for AP was evaluated to be 2.23 mmol/L. The E. coli-based biosensor has detection limits of 5.58 × 10−11 mol/L for mercury ion, 5.10 × 10−10 mol/L for cadmium ion, and 1.38 × 10−9 mol/L for zinc ion. The prepared biosensor can respond steadily for seven days.