Handheld biosensors have attracted substantial attention for numerous applications, including disease diagnosis, drug dosage monitoring, and environmental sensing. This study presents a novel handheld biosensor based on a gradient grating period guided-mode resonance (GGP-GMR) sensor. Unlike conventional GMR sensors, the proposed sensor’s grating period varies along the device length; hence, the resonant wavelength varies linearly along the device length. If a GGP-GMR sensor is illuminated with a narrow band of light at normal incidence, the light resonates and reflects at a specific period but transmits at other periods; this can be observed as a dark band by using a complementary metal oxide semiconductor (CMOS) underneath the sensor. The concentration of a target analyte can be determined by monitoring the shift of this dark band. We designed and fabricated a handheld device incorporating a light-emitting diode (LED) light source, the necessary optics, an optofluidic chip with an embedded GGP-GMR sensor, and a CMOS. LEDs with different beam angles and bandpass filters with different full width at half maximum values were investigated to optimize the dark band quality and improve the accuracy of the subsequent image analysis. Substrate materials with different refractive indices and waveguide thicknesses were also investigated to maximize the GGP-GMR sensor’s figure of merit. Experiments were performed to validate the proposed handheld biosensor, which achieved a limit of detection (LOD) of 1.09 × 10−3 RIU for bulk solution measurement. The sensor’s performance in the multiplexed detection of albumin and creatinine solutions at concentrations of 0–500 μg/mL and 0–10 mg/mL, respectively, was investigated; the corresponding LODs were 0.66 and 0.61 μg/mL.