A label roll positioning device is provided for a barcode printer and includes at least one slide rail, at least one positioning rail, and at least one pair of roll retainers. The slide rail and the positioning rail are arranged in a parallel manner inside a housing of the barcode printer. The positioning rail forms teeth on a surface thereof. At least one of the roll retainers has a bottom forming a mounting portion that is slidably fit and mounted to the slide rail for being slidable along the slide rail. The roll retainers are spaced to receive a roll of label therebetween in a securely held manner. The roll retainer is provided therein at least one positioning plug having an end forming a locking section. The positioning plug can be downward driven to have the locking section downward projecting out of the roll retainer, and the positioning plug can be upward driven to have the locking section back into concealment inside the roll retainer. Thus, with the roll of label received between the roll retainers, the positioning plug is downward driven and the locking section engages the teeth of the positioning rail to effect positioning and fixing for securely holding the roll of label between the roll retainers. A positioning device with movable and adjustable locking engagement can be realized.