Enzymeless electrochemical sensors have received considerable interest for the direct, sensitive, and selective monitoring of biomolecules in a complex biological environment. Here, we designed a nonenzymatic electrochemical sensor based on Mn2O3 nanolayers (NLs)/carbon (C) and Mn2O3 flower-like (FL)/C structure to detect acetylcholine (ACh) molecules in human fluids. The sensing properties and electrochemical activity varied based on the structural and chemical composition of Mn2O3-based materials. The Mn2O3NLs/C structure of two-dimensional NLs was arranged in parallel with a heterogeneous surface texture, stair-like step-by-step layer formation, and cracked layers that formed free spaces. Mn2O3FL/C was formed with an FL structure. The parallel and perpendicular buildup of sheets from the bottom to top and sheets spreading in all directions formed the FL structure of Mn2O3 with multi-structural defects and edges, and heterogeneous surface texture. This unique surface property of Mn2O3FL/C and composition facilitated target diffusion through the inner/outer surface and shortened the distance pathway. Moreover, the presence of carbon on the surface of Mn2O3 induced sensitivity and stability of Mn2O3, enhanced the electrochemical activity with high catalytic activity, hastened electron diffusion, and high loading of ACh molecules. The nonenzymatic ACh sensors of Mn2O3NLs/C and Mn2O3FL/C showed a good sensor design with low limits of detection (2 and 7 μM, respectively) and a linear range of 0.1–7 mM. The fabricated sensors provided high stability and selectivity, easy fabrication, multi-usage, and fast response motioning of ACh in a complex mixture of human fluids. The designed nonenzymatic sensors of Mn2O3NLs/C and Mn2O3FL/C signaled the ACh molecules with high stability and selectivity and can be used to investigate and follow up on several neuronal disorders.