An in situ and real-time investigation was performed using an optical cell system and in-silico analysis to reveal the impacts of pressure and temperature on the conformational state and behaviours of Rhizopus chinensis lipase (RCL). The fluorescence intensity (FI) of RCL increased remarkably under high pressure, and part of this increase was recovered after depressurization. This result displayed the partially reversible conformational change of RCL, which may be associated with the local change of Trp224 near the catalytic centre. High temperature caused a significant loss of secondary structure, whereas the α-helical segments including the lid were preserved by high pressure even at temperatures over 60 °C. The parameters of enzymatic reaction monitored by UV showed that the hydrolysis rate was remarkably enhanced by the pressure of 200 MPa. In the pressure range of 0.1–200 MPa, the active volume measured by the in situ system decreased from −2.85 to −6.73 mL/mol with the temperature increasing from 20 °C to 40 °C. The high catalytic capacity of the lipase under high pressure and high temperature was primarily attributed to pressure protection on RCL.