Aqueous two-phase extraction was used for efficient separation of biological particles with similar size, density, or other physical properties. The partitioning behavior of spores and parasporal crystals of Bacillus thuringiensi were studied in two types of aqueous two-phase systems: polymer/salt systems and polymer/polymer systems. The spores and crystals showed strong tendency towards the top polyethylene glycol (PEG)-rich phase and interface in PEG/salt systems, and were hardly influenced by several factors, such as concentration of PEG and salt, phase separation time, and phase ratio. Using the appropriate system (PEG 6000 2%/(NH4)(2)SO4 16%), collection of both spores and crystals directly from fermentation broth was carried out with a yield of 96% crystals on lab-scale. Separation of crystals from spores could be achieved using the polymer/polymer systems. It was found that phase-forming polymers, pH, and phase separation time showed significant influence on the partition of spores, and the optimum conditions were chosen as PEG 6000 6%/DEX60 7.5% system, pH 10.0, and 1.5 similar to 2.0 hr for the separation of crystals from spores. The yield of crystals was 83%, and more than 90% of spores could be removed after four cycles of extractions of the bottom phase with fresh top PEG-rich phase. Finally, a process scheme was developed that included an initial extraction of both spores and crystals using PEG/salt system and integrated with subsequent removal of spores by extraction in PEG/dextran system. The feasibility of the integrated separation process was verified by lab-scale application. The total yield of crystals could reach 81.4%, and more than 90% of spores could be removed.