Cyanobacteria bloom in territorial waters leads to the production of dangerous cyanotoxins such as Microcystin, which causes world-wide problems. We exerted an optimized PCR for detection of Microcystin-producing gene of Cyanobacteria from óAnzali Lagoon. The shallow coastal Anzali lagoon is located in the province of Gilan (a temperate region) in Northern Iran. It covers an area of about 200 km2 situated between 37°28 ì N and 49°25 ì E. It is26 km long and 2.0 – 3.5 km wide. For sampling, 20 stations were selected within western, central and eastern parts of the Lagoon. DNA extraction was performed by using DNG-Plus Kits. For two universal (23S30R, CYA106F) and two specific primers (mcyA-Cd1F, mcyA-Cd1R), PCR test was optimized, and its sensitivity and specificity were evaluated. Then the test was applied for samples from Anzali lagoon. Cloning of the two fragments was also performed. Application of the optimized PCR method to the DNA samples from Anzali Lagoon revealed that all of the stations contained Cyanobacteria, among which only ten indicated Microcystin-producing Cyanobacteria. In addition, a successful process of cloning and extraction of recombinant plasmids was achieved that its products were stored for future investigations. Anzali lagoon might be in danger of predomination of Cyanobacteria. Therefore, our optimized PCR method can be implemented as helpful tool for monitoring and controlling of Cyanobacteria, especially Microcystin-producing species in the lagoon.