The activity of the colon is regulated by chemical signaling, of which serotonin (5-HT) is a key transmitter. Monitoring of mucosal 5-HT overflow has been achieved to date using microelectrodes on a small segment of colonic tissue; however, little is known if such measurements are reflective with regards to 5-HT signaling from the entire colon. This study focused on developing an electrochemical array device that could be utilized to conduct multisite measurements of 5-HT overflow from the entire colon. A 3D printed mold was fabricated that could house 6 multiwall carbon nanotube composite electrodes and provide a fixed distance between the electrodes and the tissue along the entire length of the colon. The electrodes were assessed for sensitivity, stability, and crosstalk before conducting in vitro measurements using colons obtained from 6- and 24-month old mice. As composite electrodes can have a high degree of variability, normalization factors were required between electrodes for a given array. The device had the sensitivity and stability required for 5-HT measurements from intestinal tissue. Regio-specific changes in 5-HT overflow were observed with age, where increases in 5-HT overflow were observed in the distal colon due to an impairment/loss in the serotonin transporter (SERT). Our strategy can be utilized to develop arrays of varying sizes and geometries, which can offer practical solutions for large-scale tissue measurements.