Highly sensitive mode-multiplex miniaturized sensors enable the detection and quantification of multiple biomolecules simultaneously through their real-time interactions. Here, we demonstrate a grapefruit photonic crystal fiber (PCF)-based mode-multiplex surface plasmon resonance (SPR) sensor that detects multiple analytes simultaneously. Three grapefruit-shaped air-holes are internally coated with plasmonic gold (Au) material, which allows them to act as mode-multiplex channels that detect three unknown analytes. The sensor performance was investigated using the finite element method (FEM), and the optimized fiber structure was fabricated with the standard stack-and-draw method. For the y-polarized mode, channels one and three showed the maximum wavelength sensitivities of 2000 and 18,000 nm/RIU (refractive index unit) at the analyte refractive indices of 1.34 and 1.41, respectively. On the other hand, channel two showed the maximum wavelength sensitivity of 3000 nm/RIU at the analyte refractive index (RI) of 1.36 for the x-polarized mode. To the best of our knowledge, this is the first demonstration of a mode-multiplex grapefruit PCF-based SPR sensor to simultaneously detect and quantify three different analytes. We anticipate that the proposed sensor will find potential applications in the detection of real-time biomolecular interactions and binding affinity.