A long-standing goal has been to create artificial enzymes with natural enzyme-like catalytic activity. Herein, a laccase-mimicking catalyst (GSH-Cu) is designed by simulating the copper active sites and spatial amino acid microenvironment of natural enzymes. In particular, the engineered GSH-Cu shows a catalytic function that conforms to Michaelis-Menten kinetics of natural laccase. The high catalytic activity of GSH-Cu can be easily inhibited by thiram through surface passivation to produce copper nanoparticles. We demonstrate that the developed GSH-Cu with high stability and recyclability can be used to fabricate effective colorimetric sensor for sensitive detection of thiram. The resulting absorption intensity can be employed to quantify thiram in the range of 2.5–250 ng mL⁻¹, which meets the detection requirement in fruit. Bestowed with the feasibility analysis of colorimetric output, a portable platform is designed by integrating GSH-Cu based test paper with a conventional smartphone for conveniently on-site quantified thiram. The proposed strategy about engineering enzyme mimicking catalysts with excellent catalytic performance will open avenues for boosting the sensing application.