Prussian Blue (PB)/chitosan enzyme mimetic tubular micromotors are used here for on-the-fly (bio)-sensing assays. The micromotors are easily prepared by direct deposition of chitosan into the pores of a membrane template and in situ PB synthesis during hydrogel deposition. Under judicious pH control, PB micromotors display enzyme mimetic capabilities with three key functions on board: the autonomous oxygen bubble propulsion (with PB acting as a catalase mimic for hydrogen peroxide decomposition), 3,3',5,5'-tetramethylbenzidine (TMB) oxidation (with PB acting as a peroxidase mimic for analyte detection), and as a magnetic material (to simplify the (bio)-sensing steps). In connection with chitosan capabilities, these unique enzyme mimetic micromotors are further functionalized with acetylthiocholinesterase enzyme (ATChE) to be explored in fast inhibition assays (20 min) for the colorimetric determination of the nerve agent neostigmine, with excellent analytical performance in terms of quantification limit (0.30 μM) and concentration linear range (up to 500 μM), without compromising efficient micromotor propulsion. The new concept illustrated holds considerable potential for a myriad of (bio)-sensing applications, including forensics, where this conceptual approach remains to be explored. Micromotor-based tests to be used in crime scenes are also envisioned due to the reliable neostigmine determination in unpretreated samples.