The detection of phase separation and crystallization in biopolymer blends is an important aspect of biopolymer processing. In this study, attenuated total reflection (ATR)-Fourier transform infrared (FTIR) spectroscopic imaging has been applied to visualize the crystallization and phase separation of a poly(3-hydroxybutyrate) (PHB)/poly(l-lactic acid) (PLLA) blend simultaneously in situ. The peak shift of the nu(C-O) band of PHB during the isothermal crystallization process revealed a transformation of the intermolecular interaction between PHB and PLLA in the miscible state to the inter- and intramolecular interactions between PHBs in the immiscible state. The appearance and gradual separation of crystalline PHB-rich domains and crystalline PLLA-rich domains in the acquired ATR-FTIR spectroscopic images clearly showed the whole dynamic process of phase separation. With increasing isothermal crystallization time, the larger size of crystalline polymer-rich domains and the increasing integrated absorbance of the nu(C-O) band of crystalline polymers both revealed the increasing degree of crystallization in the polymer blend, which is constant with the isothermal crystallization process of pure polymers. The extent of phase separation between crystalline PHB-rich domains and crystalline PLLA-rich domains is enhanced with increasing isothermal crystallization temperature. This is because the higher isothermal crystallization temperature slows down the crystallization, which leaves more time for phase separation. The degree of crystallization in the blend decreased with increasing isothermal crystallization temperature, which was also constant with the isothermal crystallization process of the pure polymers. Finally, a new proposal for the mechanism of phase separation and crystallization in the polymer blend has been discussed. It was found that decreasing the isothermal crystallization temperature is an effective method to restrict the phase separation and control the final morphology in the upper critical solution temperature (UCST) crystallizable polymer blend.