[Background] Poor thermostability of Rhizopus chinensis lipase limits its potential in industrial applications. [Objective] Rationale molecular design was used to improve the thermostability of r27RCL (from Rhizopus chinensis lipase CCTCC M201021). [Methods] Seven eligible cysteine pair mutations were selected according to the prediction of the Disulfide by design algorithm. Mutant gene was produced by whole-plasmid PCR and then expressed in Pichia pastoris to obtain mutant lipases. [Results] The optimal mutant enzyme m9/10 (S85C-Q145C) showed improved thermostability with a 4.5-fold increase in t1/2 at 60 °C and a 4.2 °C increase of Tm compared with the parent enzyme, without compromising the catalytic efficiency. A disulfide bond between 85 cysteine residue (located in 2nd β-sheet) and 145 cysteine residue (located in 4th α-helix) was introduced into the lipase according to the cystal structure, leading to the improved thermostability of lipase. [Conclusion] The thermostability of fungal lipase could be greatly improved by newly introduced disulfide bond.