We report the first demonstration of electrochemical detection for paper-based microfluidic devices. Photolithography was used to make microfluidic channels on filter paper, and screen-printing technology was used to fabricate electrodes on the paper-based microfluidic devices. Screen-printed electrodes on paper were characterized using cyclic voltammetry to demonstrate the basic electrochemical performance of the system. The utility of our devices was then demonstrated with the determination of glucose, lactate, and uric acid in biological samples using oxidase enzyme (glucose oxidase, lactate oxidase, and uricase, respectively) reactions. Oxidase enzyme reactions produce H2O2 while decomposing their respective substrates, and therefore a single electrode type is needed for detection of multiple species. Selectivity of the working electrode for H2O2 was improved using Prussian Blue as a redox mediator. The determination of glucose, lactate, and uric acid in control serum samples was performed using chronoamperometry at the optimal detection potential for H2O2 (0 V versus the on-chip Ag/AgCl reference electrode). Levels of glucose and lactate in control serum samples measured using the paper devices were 4.9 +/- 0.6 and 1.2 +/- 0.2 mM (level I control sample), and 16.3 +/- 0.7 and 3.2 +/- 0.3 mM (level II control sample), respectively, and were within error of the values measured using traditional tests. This study shows the successful integration of paper-based microfluidics and electrochemical detection as an easy-to-use, inexpensive, and portable alternative for point of care monitoring.