Graph Microbial transglutaminase (MTGase) is an enzyme widely used in the food industry. In this study, MTGase had been applied to produce fresh cheese made from whole milk powder. To evaluate the impact of factors in reconstituted milk under the treatment of MTGase, a set of 18 experiments was conducted. Besides, a response surface methodology (a central composite design) was applied to evaluate the quality properties of fresh cheese according to the objective functions of hardness, yield, protein content, total solid content, and sensory evaluation score. In detail, enzyme concentration (0.6-3.0U/g protein), reaction temperature (30-60°C), and reaction time (1.5-6.0h) were three factors used in this model. The results showed that all these functions reached the optimal values at treated temperature, reaction time, and enzyme concentration of 36.14°C, 4.53h, and 2.59U/g protein, respectively. Furthermore, scanning electronic micrographs also showed that the network structure of the experimental products became more uniform under enzymatic treatment. The quality properties of fresh cheese (sensory evaluation score, syneresis, acidity, and the total number of lactic acid bacteria) met the CODEX STAN 243-2003 revised 2010 for the fermented milk products. Generally, following 28 days of storage, the quality properties of fresh cheese samples are stable.