The application of most probable number (MPN) method in counting the numbers of photosynthetic bacteria (PSB) showed that this method was suitable for PSB counting under illumination and anaerobic condition, which remedied the shortages of other methods. With this method, the sensitivity and accuracy of the counting could be enhanced when more replicated tubes and lower dilution times for fewer bacteria samples were applied. When PSB were not dominant in soil and water, MPN method could be used instead of other methods. PSB agent could only survive 6 approximately 9 days, and not be dominant in the environment in a long-term. Its application time and oxygen level affect the numbers of PSB significantly, which were the largest in a short application time and under anaerobic condition.