The effects of Ca2+ on the formation and structure of milk gel of mozzarella cheese were assessed by two-photon confocal laser scanning microscopy combined with fluorescence dyeing. The results show that Ca2+ can stimulate the formation of milk gel extremely and have important effects on the structure of the milk gel, the gel time decreases to 12min (3mmol/L Ca2+), 6min (6mmol/L Ca2+) and 3min（9mmol/L Ca2+）respectively from 38min (0mmol/L Ca2+) after adding of Ca2+. It was found by the real-time investigations that the fine casein granules of milk without Ca2+ became to move closer and combined with the crunodes nearby and form bigger clusters. While the milk with Ca2+ tends to coagulate in a very short time and form very strong gel, and this may prevent the casein granules from closing up to the crunodes, so the curds are smaller and connections of crunodes are weaker.