L-Cysteine is a common amino acid that plays an important role in human livelihood and production. Therefore, a novel method for the simultaneous quantitative and qualitative determination of L-cysteine by a colorimetric detection system is proposed. As a viable oxidase mimic, [N4444]Cl-G/MnO2, which consisted of MnO2 nanosheets functionalized by a tetrabutylammonium chloride-glycerol ([N4444]Cl-G) based deep eutectic solvent (DES) was fabricated. Owing to the oxidation of MnO2 nanosheets, [N4444]Cl-G/MnO2 could oxidize the colorless 3,3',5,5'-tetramethylbenzidine (TMB) into a blue product (oxTMB) with the characteristic UV-vis spectrum absorbance at 652 nm. The oxidation of TMB by DES/MnO2 was inhibited when L-cysteine was introduced, and the absorbance decreased proportionally with the increase in L-cysteine concentration. Due to this inhibition effect, a colorimetric detection system ([N4444]Cl-G/MnO2-TMB) was developed for the quantitative determination of L-cysteine. Under optimal conditions, the assay showed good linearity over the concentration range of 0.125-2.00 μg mL-1 with a low detection limit of 5.96 ng mL-1. A study of the inhibition mechanism demonstrated that the sulfhydryl group of L-cysteine could decompose [N4444]Cl-G/MnO2 into Mn2+, thus limiting the conversion of TMB to oxTMB. In addition, the [N4444]Cl-G/MnO2-TMB system was used in test strips for the visual qualitative detection of L-cysteine. The selectivity and test strip results demonstrated the high selectivity, simple operation, and rapid response of the [N4444]Cl-G/MnO2-TMB system for the qualitative detection of L-cysteine. Given the satisfying performance of the detection strategy, colorimetric sensing based on the [N4444]Cl-G/MnO2-TMB system is considered to have prospective application value in the quantitative and qualitative detection of L-cysteine.