The invention discloses a tear film positioning mechanism of a full-automatic backlight assembly machine. The tear film positioning mechanism comprises X-direction movement and Y-direction cylinder movement, wherein the size of glass is respectively positioned by X-direction and Y-direction, the Y-direction is positioned by the cylinder, and the cylinder is adjusted, so that the size specification of the glass is increased; double-row guide rails are arranged at the two sides of the cylinder, so that the glass is relatively stable. The tear film positioning mechanism provided by the invention has the advantages that the size of glass is positioned by X-direction and Y-direction, matched positioning guide rails are arranged at the two sides of the cylinder, the adjustable range of left and right sides is large, the cylinder is adjustable, the size of glass is increased, and the problems that the size of glass is small and the adjusting range is small are solved; meanwhile the glass is positioned conveniently and effortlessly by the arrangement of the positioning cylinder, and the cost is reduced.