To solve the long lens moving distance and lens defocusing when Laser Chemical Vapor Deposition（LCVD）equipment traces defect points in photomask defect repairing， a kind of ant colony optimization （ACO） algorithm mixed with 2-opt neighborhood search was proposed to optimize the sequence of defect coordinates. The method could effectively reduce the points tracing time and lens defocus rate， compared with the traditional ascending sequence of X or Y axis. To enhance the algorithm processing time in the case of large-scale defects， several acceleration tactics were applied in the ACO algorithm mixed with 2-opt neighborhood search， including nearest neighborhood searching tactic in ACO algorithm， fixed radius searching setting and “don’t look bits tactic” in 2-opt algorithm. The experimental results showed that the improved ACO algorithm mixed with 2-opt neighborhood search could reveal over 92.5% improvement than raw AOI distance， with only 5.72 s time consumption and 0.28% defocus rate. The improved algorithm demonstrates better performance in path optimization quality， optimization time and maintaining lens focusing than basic ACO and basic ACO mixed 2-opt algorithm.