Screen printing of cotton fabric using newly synthesized azo reactive dyes was carried out in the present study. Functional group chemistry and its effect on the printing properties of cotton fabric by varying the nature, number and position of reactive groups of synthesized azo reactive dyes (D1–D6) was studied. Different printing parameters (Temperature, alkali and urea) and their effect was explored on the physicochemical printing properties e.g., fixation, color yield, and penetration of the dyed cotton fabric. Data revealed that dyes with more reactive groups and having linear and planar structures (D-6) showed enhanced printing properties. Spectraflash spectrophotometer was used to evaluate the colorimetric properties of screen-printed cotton fabric and results showed superb color buildup. Printed cotton samples displayed excellent to very good ultraviolet protection factor (UPF). Presence of sulphonate groups and excellent fastness properties may entitle these reactive dyes as commercially viable for urea free printing of cotton fabric.