The invention relates to the technology of refractory metal surface treatment, in particular to a preparation method of an MoSi2 coating layer on the surface of niobium alloy. The preparation method comprises the following steps of firstly, sputtering an Mo-W thin layer on the surface of the niobium alloy; preparing the MoSi2 coating layer on the Mo-W thin layer by a laser cladding technology, soas to obtain the MoSi2 coating layer with excellent mechanical property and good bonding property with the niobium alloy. The prepared MoSi2 coating layer on the surface of the niobium alloy has the advantages that a multi-element alloying method is adopted, and magnetron sputtering and laser cladding technologies are adopted for preparation; by adding multiple alloy elements, an alloying functioncan be produced on the MoSi2 coating layer, and the mechanical property of the MoSi2 coating layer can be improved; by forming the Mo-W thin layer via the magnetron sputtering, the bonding strength between the niobium alloy and the MoSi2 coating layer can be improved to a certain degree, and the stripping of the MoSi2 coating layer is reduced.