The aim of this in vitro study was to evaluate the effect of laser treatment on shear bond strength of a self-adhesive flowable resin composite to human dentin. Eighty extracted sound human molar teeth were used for the study. The teeth were sectioned mesiodistally and embedded in acrylic blocks. The dentin surfaces were ground wet with 600-grit silicon carbide (SiC) paper. They were randomly divided into two preparation groups: laser (Er:YAG laser, with 12 Hz, 350 mJ energy) and control (SiC). Each group was then divided into two subgroups according to the flowable resin composite type (n = 20). A self-adhesive flowable (Vertise Flow) and a conventional flowable resin (Premise Flow) were used. Flowable resin composites were applied according to the manufacturer's recommendations using the Ultradent shear bond Teflon mold system. The bonded specimens were stored in water at 37 °C for 24 h. Shear bond strength was tested at 1 mm/min. The data were logarithmically transformed and analyzed using two-way analysis of variance and Student-Newman-Keul's test at a significance level of 0.05. The self-adhesive flowable resin showed significantly higher bond strength values to laser-prepared surfaces than to SiC-prepared surfaces (p < 0.001). The conventional flowable resin did not show such differences (p = 0.224). While there was a significant difference between the two flowable resin composites in SiC-prepared surfaces (p < 0.001), no significant difference was detected in laser-prepared surfaces (p = 0.053). The bond strength of a self-adhesive flowable resin composite differs according to the type of dentin surface preparation. Laser treatment increased the dentin bonding values of the self-adhesive flowable resin.