This paper demonstrates the use of a smartphone-based sensor for fluorescence polarization (FP) analysis of biomolecules. The FP detection can rapidly sense ligand-analyte bindings by measuring molecule mobility, and thus, FP-based assays have been widely used for rapid diagnostics in clinics. Here, we implemented the FP detection apparatus using a 3D-printed compact holder and the built-in camera of a smartphone. The system offers accurate measurements of the degree of polarization by simultaneously detecting the fluorescence intensities parallel and perpendicular to the polarization of the excitation. The fluorescence signal of the sample is excited by a laser or light-emitting diode and separated by a polarization beam cube depending on the polarization. Parallel and perpendicular polarized emissions are projected onto two different regions of the sensor chip in the smartphone camera. A custom software app was developed to count the average intensity in the areas of interest and compute the degree of polarization. We validated the system by measuring the polarization of dye molecules dissolved in solutions with different viscosities. As an example of biomolecule sensing, a competitive FP immunoassay of Prostaglandin E was demonstrated using the developed system and exhibited the limit of detection of 1.57 ng/mL. The smartphone-based FP assay platform can also be implemented for the detection of toxins, disease biomarkers, and pathogens in resource-limited settings.