Active fluorescence measurements can provide rapid, non‐intrusive estimates of phytoplankton primary production at high spatial and temporal resolution, but there is uncertainty in converting from electrons to ecologically relevant rates of CO2 assimilation. In this study, we examine the light‐dependent rates of photosynthetic electron transport and 13C‐uptake in the Atlantic sector of the Southern Ocean to derive a conversion factor for both winter (July 2015–August 2015) and summer (December 2015–February 2016). The results revealed significant seasonal differences in the light‐saturated chlorophyll specific rate of 13C‐uptake, (
PmaxnormalB), with mean summer values 2.3 times higher than mean winter values, and the light limited chlorophyll specific efficiency, (αB), with mean values 2.7 times higher in summer than in winter. Similar patterns were observed in the light‐saturated photosynthetic electron transport rates (
ETRmaxRCII, 1.5 times higher in summer) and light limited photosynthetic electron transport efficiency (αRCII, 1.3 times higher in summer). The conversion factor between carbon and electrons (Φe:C (mol e− mol C−1)) was derived utilizing in situ measurements of the chlorophyll‐normalized number of reaction centers (nRCII), resulting in a mean summer Φe:C which was ∼ 3 times lower than the mean winter Φe:C. Empirical relationships were established between Φe:C, light and NPQ, however they were not consistent across locations or seasons. The seasonal decoupling of Φe:C is the result of differences in Ek‐dependent and Ek‐independent variability, which require new modelling approaches and improvements to bio‐optical techniques to account for these inter‐seasonal differences in both taxonomy and environmental mean conditions.