Single-molecule detection of nucleic acids in body fluids is vital but challenging. This work presents an optical microfiber biosensor with a metal-semiconductor-2D material hybrid nanointerface for single-molecule amplification-free detection of nucleic acids in complex body fluids. By optimizing the nanointerface components, we achieved significant enhancement of the evanescent field, enabling ultrahigh sensitivity at the microfiber surface. It allowed for the detection of DNA molecules at the single-molecule level and could identify single-base-pair mismatches. Utilizing a microscale diameter and millimeter-length design, the biosensor overcomes the limitations associated with nanosensors, providing a practical solution for point-of-care diagnostics. The sensor demonstrated its potential through ultrasensitive detection of HIV nucleic acids in body fluids such as serum, sweat, and saliva. This advancement marks a critical step forward in nucleic acid detection, facilitating early disease diagnosis, personalized medicine, and fundamental biological research, despite challenges posed by the nanosize, chain-like morphology, and environmental interference of nucleic acids.