Chlorophyll is a well-recognised essential ocean variable that is important for habitat, biogeochemical and climate modelling, yet it is difficult to obtain accurately over broad areas and synoptic timescales. While extracted chlorophyll analysed by High Performance Liquid Chromatography (HPLC) remains the gold standard, ocean gliders provide estimates of chlorophyll at high resolution by measuring fluorescence over large spatial regions. However, chlorophyll fluorescence is known to be highly variable depending on factors such as the time of day, phytoplankton species composition and their physiological state. Over a decade of operating the IMOS Ocean Glider program, we present our understanding of chlorophyll fluorescence from Ecopuck instruments in Teledyne Slocum gliders. We show the stability of the instrument over time between factory calibrations, using gain settings and scale factors. We demonstrate the highly variable fluorescent response by different phytoplankton species, and we show strong positive correlations between Ecopuck chlorophyll fluorescence estimates and those analysed by HPLC taken during 3 research cruises using 3 different gliders and Ecopucks. Finally, we recommend a correction factor for Ecopuck fluorescence data like that found in a global comparison study between these sensors and HPLC chlorophyll.