Flow-through reactors are commonly used to control and optimize reagent delivery and product removal. Although recent research suggests that transcription reactions using picogram quantities of cDNA produce RNA efficiently in a flow-through microreactor, there has not been a detailed study on the mass transport and reagent dependence of microfluidic transcription reactions. We present a novel microreactor that contains H5 influenza cDNA immobilized directly onto the reactor walls to study the kinetics and reagent dependence of in vitro transcription reactions on a microfluidic platform. Enzyme and the rNTP substrate continuously flow over the cDNA and create RNA, which flows to a downstream collection well. Using nanogram quantities of cDNA, we found that enzyme limiting conditions caused by the concentration of cDNA in a small-volume microreactor channel may be partially overcome as the enzyme binds and concentrates near the channel wall. Kinetics confirm this phenomenon and show that the timescale for enzyme binding can be approximated by t(f) = cDNA/Q[E]. Surprisingly, on-chip transcription reactions have a strong dependence on the rNTP concentration from 5 to 9 mM despite a low consumption rate of rNTP molecules that is largely independent of the flow rate. Faster flow rates decrease the time it takes to fill DNA promoter sites with enzyme while additionally refreshing rNTP and MgCl2 to allow for a greater consumption of rNTP. These two effects cause reactions with higher concentrations of cDNA in the reactor channel to have a greater dependence on the flow rate. At high flow rates (> 0.37 nL/s), the reaction rate begins to drop, likely because of the release and escape of enzyme molecules from the cDNA layer. This critical flow rate can be predicted by a new modified Peclet number, Pe(m) = LcV/D, where Le is the full length of the tightly packed cDNA molecules, V is the velocity at the DNA/fluid interface, and D is the diffusivity of the enzyme molecule. Together, these insights can inspire reactor designs for a variety of applications.