In the present study, the effects of the different molecular weight organic substrates on the sludge granulation were investigated. The operation of the reactor was combined with the microfluidic chip technology to analyze the relationship between the microscopic phenomena, physicochemical properties of the granular sludge, and reactor performance in the process of sludge granulation. The use of starch, which has flocculation properties, and sodium glutamate as carbon source contributed to good settleability. The high permeability of sodium acetate and sodium propionate led to fluffy granules with an unstable settleability but a rapid size growth. From the microfluidic experiments, it was found that the sludge flocs secreted yellowish substances, which increased the overall floc volume and promoted mutual adhesion between the individual flocs. The changes in the PN/PS and Zeta potential were consistent with the trend in the sludge volume in the microfluid. The extracellular polymeric substances, and other secretions containing new cells of sludge, reduced the surface potential to enhance the flocculation process and were the key products of the conversion into aerobic granular sludge.