A microfluidic paper-based analytical device (μPAD) coupled with colorimetric detection was developed for simultaneous determination of urinary oxalate, citrate and uric acid (UA) which are important biomarkers of nephrolithiasis or kidney stones. The colorimetric detections were based on enzymatic reactions using oxalate oxidase and uricase for oxalate and UA, respectively, while an indicator displacement assay (IDA) using a copper murexide complex was applied for citrate detection. The developed μPAD was successfully applied for simultaneous determination of the three biomarkers in urine within 25 min, with linear ranges of 2–40, 5–150, and 5–45 mg L⁻¹ and detection limits of 0.6, 2.9 and 3.1 mg L⁻¹ for oxalate, UA, and citrate, respectively. The values of the percent relative standard deviation (% RSD) were lower than 6.4% for inter-day and intraday measurements of oxalate, citrate and UA standards spiked in urine samples with recovery percentages in the range of 81.0–109.8%, indicating acceptable accuracy and precision of the developed method for determination of the three biomarkers in urine samples. Accordingly, the developed μPAD holds great promise to be a simple, fast, inexpensive, low-sample and reagent volume, reliable and portable tool for simultaneous determination of oxalate, citrate and UA in urine, especially for on-site analysis.