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Magnetic labels are one of the most important components in giant magnetoresistance (GMR) biodetection systems and affect detection signals directly. Many researchers have improved the detection performance of GMR biosensors by optimizing the properties of their magnetic labels, including size, shape, coercivity, and magnetization. In most studies, magnetic labels are usually spherical, and there is little research involving alternative shapes. In this work, we prepared spindlelike Fe3O4 nanoparticles (NPs) by hydrothermal and heating reduction. The as-prepared magnetic NPs were incorporated in the GMR biodetection system. An ultralow limitation of detection concentration of 0.05 ng/ml was achieved, which can be attributed to the shape anisotropy of the spindlelike magnetic particles. A wide linear work range, 0.05–1000 ng/ml, can be achieved by the use of the spindlelike Fe3O4 NPs in GMR biodetection. Moreover, the NPs exhibit good repeatability after multiple measurements, which can be attributed to the NPs’ stable and effective magnetic diffusion field. The use of spindlelike Fe3O4 NPs as magnetic labels in this work provides a new method for improving the sensitivity and repeatability of GMR biodetection.