A novel blue-emitting fluorescent alginate derivative has been successfully synthesised in a simple two-reaction step procedure, using an aqueous conjugation strategy that involved carbodiimide coupling followed by an alkyne-azide "click" reaction. The modified alginate maintained the characteristic ability to form mechanically stable hydrogels by ionic crosslinking. The fluorescent properties of the developed biomaterial were investigated both in solution and hydrogel states, revealing that grafting of the coumarin fluorophore to alginate greatly enhanced its fluorescent properties. Importantly, hydrogels maintained around 80% of their initial fluorescence upon long periods of incubation under physiologic conditions. The fluorescent alginate hydrogels showed to be biocompatible in vitro, supporting the viability, metabolic activity and proliferation of mammary epithelial cells and, more importantly, their morphogenesis into spheroids and polarized acini-like structures. These hydrogels were further applied in the establishment of cell-in-gel microarrays for high-throughput screening of cell behaviour in three-dimensional (3D) matrices, being essential for spotting optimization and analysis. Collectively, our results highlight the potential of coumarin-grafted blue-emitting fluorescent alginate as a valuable tool for biomedical applications where hydrogel tracing is required.