With the increase awareness for a healthier food regime and greener environmental processes, microalgae are being looked as a solution for a sustainable production of polyunsaturated fatty acids, such as omega-3 eicosapentaenoic acid (EPA). Nannochloropsis oceanica is an oleaginous microalga, well-known for the ability of EPA accumulation, although higher lipid productivities are still required to make the process competitive. Therefore, three cultivation parameters were tested in the present work (temperature, light cycles and nitrogen supply) in order to study the EPA profile in the polar and neutral fractions of the cells. In addition, an online monitoring tool based on a fluorescence spectroscopy technique was developed with the aim of increasing process knowledge at real time. The results of this work show that nitrogen depletion induces the highest variability in EPA accumulation in the neutral fraction (triacylglycerols). However, to increase the EPA content in the polar fraction a different strategy needs to be implemented, such as decreasing the cultivation temperature or the light available per cell. Chemometric models were developed through PCA (Principal Component Analysis) and PLS (Projection to Latent Structures), using only fluorescence spectra as inputs, enabling the monitoring of EPA in both fractions separately. High explained variance was observed (above 85%) in both fractions, with R2 above 0.81 and slopes above 0.93 for both validation and training data sets. Lower values of cross-validation and prediction errors were observed (between 0.29 and 0.49% g/gDW). The results obtained show that fluorescence spectroscopy is a powerful technique for online monitoring of non-fluorophore molecules, such as EPA, in complex process like microalgae cultivation.