The method of loading Ca²⁺ fluorescent probe (Fluo4/AM) in pollen tube under vacuum infiltration was studied with Fengshui pear pollens which were cultured for 2 h. The results showed that: (1) pollen germination rate and activity of pollen tube were not affected by vacuum infiltration treatment for 2 hours. (2) The pollen tubes cultured for 2 h were subjected to vacuum infiltration under different conditions to assist Fluo4/AM probe to enter the pollen tubes. Laser confocal microscopy showed that Ca²⁺ gradient could be observed at the tip of pollen tube at low temperature (4 ℃) under vacuum infiltration (-80 kPa) loading for 30 min. (3) The fluorescence density of pollen tube was also significantly decreased by inhibiting the external Ca²⁺ influx or decreasing the external Ca²⁺ concentration of pollen tube. The study suggests that the method of vacuum infiltration auxiliary loading can effectively promote the entry of fluorescent probes into pollen tube cells and the binding of Ca²⁺.