Microbial co-cultures provide significant advantages over commonly used axenic cultures in biotechnological processes, including increased productivity and access to novel natural products. However, differentiated quantification of the microorganisms in co-cultures remains challenging using conventional measurement techniques. To address this, a fluorescence-based approach was developed to enable the differentiated online monitoring of microbial growth in co-cultures. Co-cultures of Streptomyces species (Streptomyces sp./spp.) with Trichoderma reesei (T. reesei) were investigated. T. reesei was tagged with the fluorescent protein mCherry, while Streptomyces spp. were used as untagged wild-type strains. First, two-dimensional (2D) fluorescence spectra of the individual microorganisms were recorded using an in-house-built device to identify characteristic fluorescence areas for online monitoring of Streptomyces spp. These spectra revealed areas of strong autofluorescence for Streptomyces spp., while T. reesei exhibited little to no autofluorescence, making these areas promising for differentiating the microorganisms in co-cultures. Time-resolved measurements further refined these results, identifying the wavelength combination 405/580 nm as particularly useful for online monitoring the growth of Streptomyces spp. To online monitor the co-culture, autofluorescence for Streptomyces spp. and mCherry fluorescence for T. reesei were used. This approach allowed for differentiated online monitoring of the co-cultures, confirmed by microscopic images. Additionally, the developed method was applied to screen varying inoculation ratios of the microorganisms in co-culture, revealing dynamic changes in the co-culture composition. This highly promising method enables reliable, differentiated online monitoring of co-cultures and has the capacity for expansion to other species, making further validation essential to confirm its potential.