This study evaluated how light spectrum, salinity, and glucose level affect Spirulina morphology, using response surface methodology (RSM). A full factorial experiment was conducted including three light conditions with different predominant spectral characteristics (“white”: 100% white; “red”: 80% red, 15% blue, and 5% green; and “yellow”: 80% yellow, 15% blue, and 5% green), four NaCl concentrations (0, 5, 15, 30 g/L), and four glucose levels (0, 1.5, 2, 2.5 g/L). Light spectrum conditions and salinity had significant effects on the filament length, spiral diameter, and screw pitch length. Glucose only had a significant effect in terms of the spiral diameter. The maximum filament length (1274 μm; under 30 g/L NaCl and 2.5 g/L glucose), spiral diameter (47 μm; under 1 g/L NaCl and 2 g/L glucose), and screw pitch length (112 μm; under 1 g/L NaCl and 2 g/L glucose) were attained under yellow light. In line with the simulation, more compacted filaments, that is, with a greater diameter (41 μm) and shorter screw pitches (78 μm), are observed under “red” light (4 g/L NaCl and 2.5 g/L glucose). Because longer but compact filaments are desirable for easier harvesting, we recommend a light spectrum that is predominantly red with a NaCl concentration of 5.8 g/L deprived of glucose, for the cost-effective harvesting of Spirulina biomass.