In the present study, a screen-printed electrode based-sensor with electrochemical detection was developed for rapid and sensitive determination of acetylcholine. At first, the screen-printed carbon electrode was modified by using a magnetic core shell and then was used for voltammetric determination of acetylcholine in ampoule, serum and urine samples. The electrochemical behaviour of acetylcholine at the modified electrode was investigated by cyclic voltammetry. The modified electrode displayed a decrease in the overpotential (ca. 130 mV) and an obvious increase in the peak current compared to the non-modified screen-printed electrode. The results indicated that modified screenprinted electrode enhanced electrocatalytic activity towards the oxidation of acetylcholine. Under optimized conditions, the limit of detection from the experiment of acetylcholine determination was 0.02 µM with acetylcholine concentration in range of 0.1-500.0 µM. The reproducibility of the measurements was tested by recording the responses for 50.0 µM acetylcholine with four different developed sensors prepared on the same manner and the same day. A relative standard deviation value of 4.3% was obtained. Finally, a recovery test is done as a part of accuracy evaluation of the system. which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.