Our previous work has demonstrated that ultrafiltration has the potential for the isolation of photosystem Ⅰ(PSⅠ) from spinach at a large scale. In order to develop a practical bioseparation process, the “product” and the “impurity” should be characterized in order to choose suitable membrane and optimize the operational and physiochemical parameters. One easily adjusted but critically important parameter, the solution ionic strength, and its effects on the properties of target protein (PSⅠ) and the major impurity (photosystem Ⅱ, PSⅡ) were studied. Following the isolation of PSⅠ and PSⅡ from spinach, the aggregation size, zeta potential, atomic force microscopy imaging and thermal stability were determined. Solution ionic strength was found to have a significant influence on the activity, aggregation size and thermal stability of both PSⅠ and PSⅡ. The results also demonstrated that the “molecular sieve effect” played a central role in the newly developed ultrafiltration process for PSⅠ isolation.