Adenosine triphosphate (ATP) bioluminescence has been widely used for biosensing applications. Commercially available platforms to perform biological testing are expensive, bulky, and require substantial amounts of reactants. Here, we report design, fabrication and testing of low cost 3D printed microfluidic chips coupled with silicon photomultipliers (SiPMs) for high sensitive real-time ATP detection. Bioluminescence current increases with increasing ATP concentration in the monitored range. The system is very sensitive to ATP concentration changes regulated by the flow rate. It exhibits a sensitivity of 1.82· 10-2 A/M and a LoD of 8 nM. Static and dynamic performance of the SiPM for ATP bioluminescence detection were evaluated by measuring different ATP concentrations. They were carried out coupling SiPM with a glass cuvette and a 3D-chip as sample holders, respectively. Using 3D-chip and two programmable syringe pumps, several ATP concentrations could be measured in continuous-flow mode by simply changing the flow-rate ratio of the reactants (ATP and standard reaction solution). The ability of the SiPM to detect the weak bioluminescence signals emitted by low ATP concentration. To demonstrate the sensitivity and the improved performance of this SiPM-based system, same ATP concentrations were measured and compared with a commercial reader (PerkinElmer Victor 2030). The calibration curve comparison, demonstrates as all systems compared exhibit similar sensitivities within the experimental errors. The combination of SiPM with 3D-chips provides a means of creating compact, sensitive and low-cost bioluminescence …