Within the framework of the semiclassical Boltzmann-Uehling-Uhlenbeck transport model,we investigate the effects of symmetry energy on the sub-threshold pion using the isospin MDI interaction with the stiff and soft symmetry energies in the central collision of 48Ca+48Ca at the incident beam energies of 100,150,200,250 and 300 MeV/nucleon.We find that the ratio ofπ- /π+ of sub-threshold charged pion production is greatly sensitive to the symmetry energy,particularly around 100 MeV/nucleon energies.Large sensitivity of sub-threshold charged pion production to nuclear symmetry energy may reduce uncertainties of probing nuclear symmetry energy via heavy-ion collision.