In order to establish an efficiency regeneration system via isolated microspore culture from single donor plant for future application in creation and screening of mutant population, each barley plant grown in climate chamber was used as single donor for isolated microspore culture, and the effects of different culture batches and different donor origins on the callus induction and plant regeneration, as well as the role of salt stress (500 mg·L~(-1) NaCl) in the microspore culture were studied. The results showed that the callus production fluctuated between the culture batches in continuous microspore culture from single donor plant, which reached the summit at the fifteenth week, and in average 2 035 plants was regenerated from single donor plant. In addition, salt stress severely repressed the callus induction and plant regeneration, at 46% and 67% decline comparing to control respectively, and the plant regeneration frequency varied between donor plants. Conclusion: It is applicable to produce homogenous plants with high efficiency from a single donor plant grown in climate chamber via continuous microspore culture, and salt stress is effective for screening during microspore culture.