Abstract: This experiment was conducted to evaluate the combined effect of ethylenediaminetetraacetic acid (EDTA) and microbial phytase on the concentration of minerals of serum and parameters of mineralization of tibia in Hy-line commercial layers (W-36) in 53-64 weeks of age. One hundred and ninty two of laying hens were tested. The experimental design was completely randomized design with a 3x2 factorial arrangement with three levels (0, 0.1 and 0.2%) of EDTA and two levels (0.0 and 300 FTU kg-1) of microbial phytase in low available phosphorus diets with 6 treatments, 4 replicates and 8 hens in each replicate. The concentration of zinc, cupper and manganese of serum and ash, calcium and phosphorus of tibia was evaluated. The results showed that adding of EDTA into low available phosphorus diets significantly affected the concentration of serum zinc and tibia ash calcium (p < 0.05). Interaction between EDTA and microbial phytase significantly affected the concentration of copper and manganese in serum and tibia ash percentage (p < 0.01). Using 300 FTU kg-1 microbial phytase into low available phosphorus diets increased tibia ash phosphorus percentage (p < 0.01).