Bioactive glass (BG) with uniform spherical morphology was prepared by EISA (Evaporation Induced Self Assembly) process using non-ionic Pluronic F127 as structure directing agent and phosphoric acid as a source of P2O5 and compared with conventional precursor triethyl phosphate (TEP). EISA method proceeds through ionic interaction mechanism by protonation of surfactant and silicic species. H3PO4 provides H+ and PO43 − ions in reaction medium which play critical role in reaction mechanism. Hydrogen ions increase the protonation of H2O and surfactant, while PO43 − ions act as bridging molecule between different cations, ensuring incorporation of phosphorus in BG network. TEP participates by proton acceptance mechanism, creating competitive environment. Thus H3PO4 facilitates the formation of BG in presence of non-ionic surfactant Pluronic F127. The prepared glasses were characterized by FTIR, SEM-EDX, TGA-DSC and BET surface analyzer. Uniform spherical morphology, improved dispersity, relatively large surface area and better cells focal attachment were observed for BG-H3PO4, prepared by using H3PO4. SiO2–CaO–P2O5 mol% composition of BG-H3PO4 was (66:24:10) close to the theoretical value (65:25:10), while for BG-TEP the actual ratio was (77:20.5:2.5). The surface reactivity, studied by soaking in simulated body fluid, showed rapid growth of hydroxyapatite with Ca/P ratio 1.67 on BG-H3PO4. The proliferation of MC3T3 cells on BG-H3PO4 was remarkably improved as compared to conventional BG. Thus BG-H3PO4 can be considered for biomedical applications in future especially for drug loading and composite application where homogeneous and uniform structure are of utmost importance.