A novel screen-printed microfluidic paper-based analytical device with all-carbon electrode-enabled electrochemical assay (SP-ACE-EC-μPAD) has been developed. The fabrication of these devices involved wax screen-printing, which was simple, low-cost and energy-efficient. The working, counter and reference electrodes were screen-printed using carbon ink on the patterned paper devices. Different wax screen-printing processes were examined and optimized, which led to an improved method with a shorter heating time (~5 s) and a lower heating temperature (75 °C). Different printing screens were examined, with a 300-mesh polyester screen yielding the highest quality wax screen-prints. The carbon electrodes were screen-printed on the μPADs and then examined using cyclic voltammetry. The analytical performance of the SP-ACE-EC-μPADs for the detection of glucose and uric acid in standard solutions was investigated. The results were reproducible, with a linear relationship [R2 = 0.9987 (glucose) or 0.9997 (uric acid)] within the concentration range of interest, and with detection limits as low as 0.35 mM (glucose) and 0.08 mM (uric acid). To determine the clinical utility of the μPADs, chronoamperometry was used to analyze glucose and uric acid in real urine samples using the standard addition method. Our devices were able to detect the analytes of interest in complex real-world biological samples, and have the potential for use in a wide variety of applications.