Anaerobic granules are responsible for organic degradation and biogas production in a reactor. The biogas production is entirely dependent on a mass transfer mechanism, but so far, the fundamental understanding remains poor due to the covered surface of the reactor. The study aimed at investigating the fundamental mass transfer characteristics of single anaerobic granules of different sizes using microscopic imaging and analytical monitoring under single and different organic loadings. The experiment was conducted in a micro reactor and mass transfer was calculated using modified Fick’s law. Scanning electron microscopy was applied to observe biogas production zones in the granule, and a lab-scale microscope equipped with a camera revealed the biogas bubble detachment process in the micro reactor for the first time. In this experiment, the granule size was 1.32, 1.47, and 1.75 mm, but 1.75 mm granules were chosen for further investigation due to their large size. The results revealed that biogas production rates for 1.75 mm granules at initial Chemical Oxygen Demand (COD) 586, 1700, and 6700 mg/L were 0.0108, 0.0236, and 0.1007 m3/kg COD, respectively; whereas the mass transfer rates were calculated as 1.83 × 10−12, 5.30 × 10−12, and 2.08 × 10−11 mg/s. It was concluded that higher organic loading and large granules enhance the mass transfer inside the reactor. Thus, large granules should be preferred in the granule-based reactor to enhance biogas production.