Using a laser-scribed (direct printing) technique, we have fabricated an enzymeless, mediatorless, and paper-interfaced electrochemical device (P-LSG) for uric acid detection on a flexible polyimide sheet. Various paper substrates were investigated, and it was found that Whatman filter paper-1 is promising to obtain the best electrochemical signals at the small volume of electrolyte, i.e., 20 μL. Furthermore, the Whatman filter paper-1 was modified with gold nanoparticles (AuNPs) to improve the electrocatalytic activity of the P-LSG device. The fabricated AuNP-modified P-LSG biosensor exhibited excellent electrocatalytic activity for the detection of uric acid over a wide range of 10 to 750 μM, with sensitivity of ∼0.214 μA μM cm, and a limit of detection of ∼1.4 μM. The sensor was further validated by using the UHPLC-ESI-MS/MS technique, and the observed percentage recovery was less than 10%. This work opens the window to modified paper substrates with various nanomaterials to improve the sensing parameters. The electrolyte storage capacity and rich chemistry of paper additionally provide an efficient immobilization platform for biorecognition elements to diagnose other metabolites. Furthermore, it has the potential to analyze the volume of small samples (like sweat, tears, urine, etc.) using paper to develop noninvasive wearable biosensors.