An analytical platform was developed using agarose gel to overcome the issues of device miniaturization and interference effects caused by foreign proteins, which are challenges for field analysis-oriented measurement technology. The lab-on-gel (LoG) device integrates sample preparation, reaction, and detection on a chip, with an agarose gel for separation that preferentially allows only small molecules to pass through due to the size exclusion effect, and an agarose gel for sensing that contains glucose dehydrogenase and substrates. Measurements can be performed simply by dropping in a diluted serum sample and then placing the device in a custom-made compact Raspberry Pi-based fluorescence imager. This analytical approach was demonstrated by glucose measurement. The detection limit for glucose was estimated to be 21 μM (3σ). The method was also able to quantify glucose in serum at a lower dilution than the conventional method using well plates. This suggests that agarose gel works effectively for the pretreatment and sensing of biosensors. The developed method is expected to be useful for rapid and labor-saving medical diagnostics in town clinics and at the bedside.