A novel label-free optical fiber biosensor, based on a microcavity fiber Mach–Zehnder interferometer, was developed and practically demonstrated for DNA detection. The biosensor was fabricated using offset splicing standard communication single-mode fibers (SMFs). The light path of the sensor was influenced by the liquid sample in the offset open cavity. In the experiment, a high sensitivity of −17,905 nm/RIU was achieved in the refractive index (RI) measurement. On this basis, the probe DNA (pDNA) was immobilized onto the sensor’s surface using APTES, enabling real-time monitoring of captured complementary DNA (cDNA) samples. The experimental results demonstrate that the biosensor exhibited a high sensitivity of 0.32 nm/fM and a limit of detection of 48.9 aM. Meanwhile, the sensor has highly repeatable and specific performance. This work reports an easy-to-manufacture, ultrasensitive, and label-free DNA biosensor, which has significant potential applications in medical diagnostics, bioengineering, gene identification, environmental science, and other biological fields.