Two different bioactive glasses, 45S5 (45SiO2–24.5CaO–24.5Na2O–6P2O5) and S53P4 (53SiO2–20CaO–23Na2O–4P2O5), were synthesized by the sol–gel method using dual-templating methods. Polymethyl methacrylate (PMMA) colloidal crystal used as a template component produced three-dimensionally ordered macroporous (3DOM) structure. Block copolymer, P123, used as a structure-directing agent and template component yielded either mesopores after calcination. The morphology of the 3DOM-mesoporous bioactive glasses (3DOM-MBGs) was observed using a scanning electron microscope. Additionally, energy dispersive spectrometer and inductively coupled plasma-optical emission spectrometry (ICP-OES) were used to confirm the elemental compositions of the samples. Specific surface area and mesopore size distribution were determined using the N2 adsorption–desorption method, where S53P4 glasses exhibited well-ordered mesoporous properties compared to 45S5 glasses. In vitro bioactivity test was performed by soaking the 3DOM-MBGs in a simulated body fluid at 37 °C for 7 days. Results indicated that 45S5 exhibited the ability to grow hydroxyapatite-like layer on the surfaces faster than S53P4.