Serotonin (5-HT) is a key signaling molecule within the mucosal epithelium of the intestinal wall and has been shown to be an important modulator of motility. At present, no single approach has been established for simultaneous dual measurement of 5-HT overflow and circular muscle contraction. We developed a 3D-printed carbon black/polylactic acid (PLA) electrochemical sensor, which had a geometry suitable for ex vivo measurement in the anorectum. The device was characterized for sensitivity and stability for 5-HT measurements as well as suitability for accurate tracking of anorectal contractions. The 3D-printed electrochemical sensor had a linear range in physiological concentrations of 5-HT (1-10 μM) present within the intestinal tract and a limit of detection of 540 nM. The sensor was stable for 5-HT measurement following ex vivo tissue measurements. There was a signficant correlation in the amplitude and duration of individual contractions when comparing the measurements using an isometric force transducer and 3D-printed electrochemical sensor. Finally, in the presence of 1 μM fluoxetine, the sensor was able to monitor a reduction in contractility as well as an increase in 5-HT overflow as predicted. Overall, the 3D-printed sensor has the ability to conduct dual simultaneous measurements of 5-HT overflow and contractility. This single device will have significant potential for clinical measurements of anorectum function and signaling that can direct therapeutic management of patients with bowel disorders.