Microalgae have been considered great candidates for carbon reduction and sustainable feedstock for foods, biofuels, and biochemicals, but their production usually features low productivity, high costs, and intensive energy inputs. The thick culture layer adopted in conventional cultivation systems (5.0−30 cm) has been established as a significant reason for the above problems. In this study, a novel mixing-based thin-layer fountain photobioreactor (TLF-PBR) was developed. A fountain pump was used to pump and spray microalgal culture for mixing. The results showed that the mixing mode could support sufficient mixing to efficiently cultivate Spirulina sp. in a 50 cm-diameter TLF-PBR, with the lowest mixing time of 13.580 ± 0.522 s, the highest oxygen mass transfer coefficient of 142.555 ± 5.791 h −1 , and the highest biomass concentration of 3.118 ± 0.009 g L −1 in the 1.0 cm layer. The TLF-PBR was successfully scaled to a diameter of 1.5 m (1.766 m 2 ), with a maximum biomass density of 3.955 ± 0.037 g L −1 , which was 71.0 and 44.7% higher than that of the flat panel PBR and thin-layer cascade system, respectively. This study provides a novel approach for developing thin-layer, scalable PBRs that could support cost-effective, efficient microalgae production.