This study was conducted to evaluate the effect of CO2 fertilization in winter plastic houses on cut flower yield and growth characteristics of Alstroemeria. The cultivars used in the experiment were Alstroemeria ‘Hanhera’ and ‘HappyAls’, and they were planted in the ground on August 25, 2021. The untreated greenhouse was maintained at a CO2 atmospheric concentration of 400 ppm, and the CO2-fertilized greenhouse was fertilized at an average concentration of 800 ppm. The concentration of CO2 reached the 800 ppm at 8 am and decreased to approximately 300 ppm by 11 am, before the side windows were opened in the CO2-treated greenhouse on January 26, 2022. This indicated that photosynthesis in Alstroemeria was most active from 8 am to 11 am. The cut flower yield of Alstroemeria ‘Hanhera’ increased by approximately 62% in the CO2-fertilized greenhouse compared to that in the untreated greenhouse. The flowering shoot height of Alstroemeria ‘Hanhera’ was 93.5 cm in the treated greenhouse, which was approximately 43% higher than that in the untreated greenhouse. These results demonstrate that CO2 fertilization during the Alstroemeria cultivation in winter plastic houses effectively increases flowering shoot height and yield.