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Mesoporous bioactive glass (MBG) has a high specific surface area, promoting the reaction area, thereby improving the bioactivity; thus, MBG is currently gaining popularity in the biomaterial field. Spray pyrolysis (SP) is a one-pot process that has the advantages of shorter process time and better particle bioactivity, therefore, MBG was prepared by SP process with various polyethylene glycol (PEG, molecular weight ranged from 2000–12,000) and acid (HCl and CH₃COOH) additions. In vitro bioactivity and mesoporous properties of the so-obtained MBG were investigated. The experimental results showed that all the MBG exhibited amorphous and mesoporous structure. Increasing the molecular weight of PEG can improve the mesoporous structure and bioactivity of MBG. Whereas optimized MBG was prepared with suitable concentration of PEG and CH₃COOH. In the present work, MBG synthesized via spray pyrolysis by adding 5 g of PEG with a molecular weight of 12,000 and 50 mL of CH₃COOH exhibited the best in vitro bioactivity and mesoporous structure.