The development of ultrasensitive, rapid, and robust point-of-care devices for biomarker detection holds great promise to revolutionize clinical diagnostics. Notably, microRNA has demonstrated significant potential as a minimally invasive cancer biomarker; however, conventional approaches for their detection require costly multistep procedures, bulky equipment, and trained personnel. Herein, we develop a peptide nucleic acid (PNA)-functionalized laser-induced graphene biosensor based on a single-step fabrication and single-step probe functionalization strategy, with a smartphone-based readout. Immobilization of a bespoke 1-pyrenebutyryl-N-end-terminated 17 mer PNA through π–π stacking on graphene was validated via Raman and X-ray photoelectron spectroscopy. As a proof-of-concept, the biosensor is applied to the detection of a prostate cancer biomarker, hsa-miR-141, exhibiting excellent analytical performance with high specificity and a limit of detection of 0.6 aM, without requiring amplification, enzymes, or thermal cycling. The versatility of this portable amplification-free platform and its ease of fabrication facilitate its translation into a point-of-care device with the potential to revolutionize clinical diagnostics, even in resource-limited settings.