Online monitoring of algal biotechnological processes still requires development to support economic sustainability. In this work, fluorescence spectroscopy coupled with chemometric modelling is studied to monitor simultaneously several compounds of interest, such as chlorophyll and fatty acids, but also the biomass as a whole (cell concentration). Fluorescence excitation-emission matrices (EEM) were acquired in experiments where different environmental growing parameters were tested, namely light regime, temperature and nitrogen (replete or deplete medium). The prediction models developed have a high R2 for the validation data set for all five parameters monitored, specifically cell concentration (0.66), chlorophyll (0.78), and fatty acid as total (0.78), saturated (0.81) and unsaturated (0.74). Regression coefficient maps of the models show the importance of the pigment region for all outputs studied, and the protein-like fluorescence region for the cell concentration. These results demonstrate for the first time the potential of fluorescence spectroscopy for in vivo and real-time monitoring of these key performance parameters during Nannochloropsis oceanica cultivation.