Difference frequency generation has long been employed for numerous applications, such as coherent light generation, sensing and imaging. Here, we demonstrate difference frequency generation down to atomic thickness in monolayer molybdenum disulfide. By mixing femtosecond optical pulses at wavelength of 406 nm with tunable pulses in the spectral range of 1300-1520 nm, we generate tunable pulses across the spectral range of 550-590 nm with frequency conversion efficiency up to ∼2 × 10-4. The second-order nonlinear optical susceptibility of monolayer molybdenum disulfide, χ, is calculated as ∼1.8 × 10-8 m V-1, comparable to the previous results demonstrated with second harmonic generation. Such a highly efficient down-conversion nonlinear optical process in two-dimensional layered materials may open new ways to their nonlinear optical applications, such as coherent light generation and amplification.