Biocompatible hydrogels are versatile platforms for encapsulating living cells in biotechnology due to their unique physical, structural and mechanical properties. The diffusion of dissolved carbon dioxide (dCO) into the hydrogel matrix is of great importance for the growth of immobilised photosynthetic cells like microalgae and cyanobacteria. However, non-invasive analysis methods for measuring the diffusion of dCO in hydrogels are limited. In this article, we describe an indirect method for the non-invasive measurement of diffusion rates for dCO in hydrogels. We visually tracked the diffusion along the axial direction of pH indicator-doped hydrogel monoliths by recording the interface position over time. We calculated the interface velocity and the pseudo diffusion coefficients (D) over time. The obtained D values are in a realistic range compared to literature values. Therefore, this novel analysis method for dCO diffusion gained valuable insights into diffusion dynamics in different hydrogels and can aid in the design of better immobilisation matrices for photosynthetic cells.•Non-invasive, rapid method for estimation of dissolved CO (dCO) diffusion in hydrogels•Automatic analysis of colour interface formation due to acidification of hydrogels by diffusing dCO•Agarose hydrogels exhibit an approximated 30x higher pseudo dCO diffusion coefficient than silica gel.