Background: Exposing goji berries to elevated CO2 (eCO2) for long periods reduces sugar and secondary metabolite contents. However, sugar accumulation in fruit depends on photosynthesis and photoassimilate partitioning. The objectives of this study were a) to explore photosynthesis, sugar content, and sucrose metabolism-related enzyme activity in goji berry leaves and fruits under ambient and elevated CO2, and b) to identify the genes encoding Lycium barbarum acid invertase (LBAI), L. barbarum sucrose synthase (LBSS), L. barbarum sucrose phosphate synthase (LBSPS), and L. barbarum neutral invertase (LBNI) based on transcriptome profiling and expression analyses in different tissues. Results: The results showed that the expression of the above genes changed significantly in plants grown under eCO2 for 90 and 120 days as photosynthetic rate increased, while leaf and fruit sugar content decreased and the activity of four sucrose metabolism-related enzymes increased in leaves, and acid and neutral invertase increased in fruits. Furthermore, a significant correlation was detected between sugar content and enzyme activity. The expression of LBAI, LBSPS, and LBNI were high in stems, whereas LBSS was predominantly expressed in fruits. Conclusions: eCO2 affects chlorophyll, photosynthesis, sugars, and related enzyme activities in goji berry leaves and fruits after 90 and 120 days of treatment. Our findings provide fundamental data on photosynthesis and sugar accumulation trends in goji berries under eCO2 exposure.