In this paper, we report the effect of the number and position of the adenosine triphosphate (ATP) and luciferase streams on the diffusion-based continuous bioluminescent reaction in a microchannel. The detectable bioluminescent reaction between substrate (ATP) and enzyme (luciferase) was maximized when the total flow rate of the substrate and the enzyme solution was around 3 and 8 mu l/min for the 2- and 3-stream configurations, respectively. Most of the light was generated inside the luciferase phase, because the diffusion of over 100 times smaller ATP than luciferase into luciferase phase is a limiting factor of continuous flow bioluminescence reaction in microchannel. The diffusion-based reaction of luciferase was enhanced by increasing the number of streams from 2 to 3. The bioluminescence was higher up to 41% when ATP solution was infused as a middle of three streams (L_A_L configuration) comparing with the A_L_A configuration. The optimized flow rates of ATP and luciferase solutions on a thick poly(dimethylsiloxane) layer were obtained as 0.15 mu l,/min:0.7 mu l/min:0.15 mu l/min for luciferase:ATP:luciferase solutions, respectively. In addition, a diffusion-based simulation model was developed for the characterization of the bioluminescent reaction in a microchannel. The numerical simulation using the model was closely matched with the experimental results with 0.01 level significant correlations in bivariate correlation coefficient analysis. The reaction between ATP and the luciferase solution in a microfluidic device can be used for a flow-type bioluminescence sensor for rapid detection of cells. Published by Elsevier B.V.