Two-dimensional (2D) metal–organic framework (MOF) nanosheets emerging as a new member of the 2D family have received significant research interest in recent years. Herein, we have successfully synthesized 2D copper-based MOF nanosheets with bimetallic anchorage using a facile two-step process at room temperature and ambient pressure, denoted as Cu(HBTC)-1/Fe₃O₄-AuNPs nanosheets. The as-synthesized 2D bimetallic MOF nanosheets displayed enhanced peroxidase-like activity with relatively high catalytic velocity and affinity for substrates compared with previously reported peroxidase mimics. Furthermore, their intrinsic peroxidase-like catalytic activity could be flexibly regulated by single-stranded DNA (ssDNA), exhibiting the enhancement of 3,3′,5,5′-tetramethylbenzidine (TMB) oxidation or inhibition of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS) oxidation due to the adsorption of ssDNA via π–π stacking. Accordingly, on the basis of their peroxidase-like activity, our prepared 2D bimetallic immobilized MOF nanosheets achieved ultra-sensitive detection of H₂O₂ with a linear range of 2.86 to 71.43 nM, and comparable detection performance for glucose with a linear range of 12.86 to 257.14 μM. By means of their controllable peroxidase-like activity, a versatile colorimetric sensing platform was developed which realized the detection of sulfadimethoxine (SDM) with a linear range of 3.57 to 357.14 μg L⁻¹ and the limit of detection (LOD) of 1.70 μg L⁻¹. With the multiplexed performance for detecting various targets, our as-synthesized bimetallic MOF nanosheets hold great promise for applications in environmental monitoring, as well as bioassays by virtue of their good biocompatibility.