Rapid and precise profiling of acetylcholine (ACh) has become important for diagnosing diseases and safeguarding health care because of its pivotal role in the central nervous system. Herein, we developed a new colorimetric sensor based on protein-inorganic hybrid nanoflowers as artificial peroxidase, comprising a test kit and a smartphone reader, which sensitively quantifies ACh in human serum. In this sensor, ACh indirectly triggered the substrate reaction with the help of a multienzyme system including acetylcholinesterase, choline oxidase, and mimic peroxidase (nanoflowers), accompanying the enhancement of absorbance intensity at 652 nm. Therefore, the multienzyme platform can be used to detect ACh via monitoring the change of the absorbance in a range from 0.0005 to 6.0 mmol L-1. It is worth mentioning that the platform was used to prepare a portable agarose gel-based kit for rapid qualitative monitoring of ACh. Coupling with ImageJ program, the image information of test kits can be transduced into the hue parameter, which provides a directly quantitative tool to identify ACh. Based on the advantages of simple operation, good selectivity, and low cost, the availability of a portable kit for point-of-care testing will achieve the needs of frequent screening and diagnostic tracking.