Real-time bioprocess monitoring is crucial for efficient operation and effective bioprocess control. Aiming to develop an online monitoring strategy for facilitating optimization, fault detection and decision-making during wastewater treatment in a photo-biological nutrient removal (photo-BNR) process, this study investigated the application of Raman spectroscopy for the quantification of total organic content (TOC), volatile fatty acids (VFAs), carbon dioxide (CO2), ammonia (NH3), nitrate (NO3), phosphate (PO4), total phosphorus (total P), polyhydroxyalkanoates (PHAs), total carbohydrates, total and volatile suspended solids (TSSs and VSSs, respectively). Specifically, partial least squares (PLS) regression models were developed to predict these parameters based on Raman spectra, and evaluated based on a full cross-validation. Through the optimization of spectral pre-processing, Raman shift regions and latent variables, 8 out of the 11 parameters that were investigated—namely TOC, VFAs, CO2, NO3, total P, PHAs, TSSs and VSSs—could be predicted with good quality by the respective Raman-based PLS calibration models, as shown by the high coefficient of determination (R2 > 90.0%) and residual prediction deviation (RPD > 5.0), and relatively low root mean square error of cross-validation. This study showed for the first time the high potential of Raman spectroscopy for the online monitoring of TOC, VFAs, CO2, NO3, total P, PHAs, TSSs and VSSs in a photo-BNR reactor.