Returning lysed sludge to biochemical tanks to achieve in-situ sludge reduction is promising. A novel approach for regulating and initiating the sludge lysis process utilizing thermophilic strains is proposed. In this study, three strains were isolated from sludge, and sequencing of the 16 S rDNA amplicon revealed that the three thermophilic strains were the closest relatives to the genus Geobacillus, and they were designated as Geobacillus sp. DX5, Geobacillus sp. DX8 and Geobacillus sp. DX11. Sludge lysis experiments demonstrated that composite thermophilic strains (CTS) were dosed at 65°C, and the sludge VSS removal efficiency after 24 h was 37.8%, significantly surpassing individual strains. Moreover, 65.8% of proteins and 52.0% of polysaccharides in Extracellular polymeric substances (EPS) of sludge were degraded. Furthermore, Three-dimensional excitation-emission matrix (3D-EEM) fluorescence spectroscopy results revealed a substantial release of dissolved organic matter during sludge lysis. Inoculation of CTS under optimal conditions (65°C, pH 7, 4 × 1010cfu/L) resulted in VSS removal efficiency and protease activity of 42.7% and 2584 U/L, respectively. After 20 days of inoculation with CTS to initiate sludge lysis reactor, VSS removal efficiency reached 46%. Additionally, it stimulated the growth of the thermophilic bacterial community, engaging indigenous thermophilic strains in the sludge lysis process. Display omitted •Three novel thermophilic bacterial strains were isolated from sludge.•Inoculated with CTS at 65 °C reduces sludge VSS by 36.8% within 24 hours.•CTS enables rapid initiation of the sludge lysis reactor.•Inoculation with CTS can rapidly adapt to the environment and colonize the sludge.