Semi-interpenetrating polymer network (semi-IPN) strategy is employed to fabricate uniform microspheres with temperature and pH dual-responsive behavior, which is composed of poly(Nisopropylacrylamide) and poly(acrylic acid) in the presence of N,N′-methylenebisacrylamide (MBA) as the cross-linker. The influences of MBA amount (M m ) on the structures and properties of the microspheres are investigated in terms of particle size, surface morphology, pH sensitivity, and thermo-sensitivity with low critical solution temperature in the range M m = 2.5-15%. Additionally, functional group distributions in the microspheres are probed by titration and employing the Henderson-Hasselbalch equation. The results show that all the properties strongly change depending on M m . Based on transmission electron microscopy and confocal laser scanning microscopy measurements, and these properties, the M m -induced changes in the structures of the semi-IPN microspheres are discussed. body, the hydrogels that respond to pH and temperature simultaneously have attracted significant attention. Poly(N-isopropylacrylamide) (PNIPAM) is one of the most extensively studied thermo-sensitive polymers, which can exhibit lowest critical solution temperature (LCST) at ≈32 °C close to human body temperature. To obtain the pH-responsive behavior of PNIPAM-based hydrogels, the formation of semi-interpenetrating polymer network (semi-IPN) has been proved to be a useful method. In 2001, Lee et al. prepared thermo-and pH-sensitive semi-IPN hydrogels composed of PNIPAM and alginate by cross-linking with calcium ions. [2] Subsequently, many efforts were devoted to prepare the pH/temperature dual-sensitive hydrogels based on PNIPAM and other polymers with semi-IPN structure, such as chitosan, poly(acrylic acid) (PAAc), and poly(aspartic acid) (PAS). [3][4][5] The resultant hydrogels exhibited improved temperature and pH responsiveness.However, the slow response rate of macroscopic semi-IPN hydrogels was still a drawback, which limited their further application in bioengineering. Considering the