Laser cladding of micro-fluidic channels mold was performed using Nd:YAG laser and synchronous powder feeder. Influences of laser power and powder feed rate on clad layer geometrical dimensions and qualities were investigated. Results show that powder feed rate 1.5-2.5 g/min used obtains micro clad layers which meet geometrical dimensions’ requirement of micro-fluidic channels mold and combination of parameters laser power 400 W and powder feed rate 2.0 g/min achieves micro clad layer with better clad quality. Scanning paths of micro-fluidic channels mold was planned through CAD-CAM software. Using optimum parameters combination, multilayer laser cladding experiment was carried out and a sample of micro-fluidic channels mold was fabricated with expectative structure and hardness. A little milling and polishing makes the sample meet technical requirements, 0.2mm in height and 0.3mm in width. Fabricating a mold takes 15-20 min totally.