A versatile guest matrix was fabricated from a temperature- and pH-sensitive poly(N-isopropylacrylamide)-co-(3-(N,N-dimethylamino)propylmethacrylamide) microgel (poly(NIPAM-co-DMAPMA), MG) for the gentle incorporation of butyrylcholinesterase (BChE). The microgel/BChE films were built up on a surface of graphite-based screen-printed electrodes (SPEs) premodified with MnO2 nanoparticles via a two-step sequential adsorption under careful temperature and pH control. On this basis, a rather simple amperometric biosensor construct was formed, which uses butyrylthiocholine as BChE substrate with subsequent MnO2-mediated thiocholine oxidation at a graphite-based SPE. The complexation of BChE with the microgel was found to be safe and effective, as confirmed by a high operational and rather good long-term storage stability of the resultant SPE-MnO2/MG/BChE biosensors. The small mesh size of the microgel with respect to the size of BChE results in a predominant outer complexation of BChE within the dangling chains of the microgel rather than a deep penetration of the enzyme into the microgels. Given such surface localization, BChE is easily accessible both for the substrate and for cholinesterase inhibitors. This was supported by the analytical characteristics of the SPE-MnO2/MG/BChE biosensor that were examined and optimized both for the substrate and for the enzyme detection. The SPE-MnO2/MG/BChE biosensor enabled precision detection of organophosphorus pesticides (diazinon(oxon), chlorpyrifos(oxon)) in aqueous samples with minimized interference from extraneous (nonanalyte) substances (e.g., ions of heavy metals). The detection limits for diazinon(oxon) and chlorpyrifos(oxon) were estimated to be as low as 6 × 10-12 M and 8 × 10-12 M, respectively, after 20 min of preincubation with these irreversible inhibitors of BChE.