The invention relates to a preparation method of an Escherichia coli (E.coli) biosensor and a product obtained thereby. The preparation method comprises the following steps: (1) knocking out three genes, namely copA, cueO and cusA in a wild-type E.coli MC4100 genome by a Red recombination system; (2) constructing a fusion reporter gene PcopA::gfpmut2 by adopting a cross PCR (Polymerase Chain Reaction) technology; (3) cloning the segment obtained in step (2) to a pMD18-T vector; (4) carrying out double digestion on a PcopA::gfpmut2-T vector to obtain a target strip, and then connecting the target strip to a pET28a vector; and (5) converting a fusion reporter vector obtained in the step (4) to a deltacopA-deltacueO-deltacusA tri-gene mutant strain obtained in the step (1). In the preparation method, the three genes such as the copA, the cueO and the cusA are deleted from wild-type E.coli MC4100, and then the genes are converted to the fusion reporter vector PcopA::gfpmut2-pET28a so as to finally obtain the product. The product obtained by the method has the characteristics of high specificity, high sensitivity, high speed, high efficiency, high cost performance and the like, and is simple and convenient to use, thus being applicable to real-time field monitoring of copper ions.