The bifunctional H-beta zeolite supported vanadium oxide catalysts were prepared at different calcination temperatures and investigated for dimethyl ether (DME) direct oxidation to polyoxymethylene dimethyl ethers (DMMx). The impact of the nature of VOx species and the acid properties controlled by the calcination temperature on the DMMx formation has been studied in detail. Vanadium oxide species dispersing well on the H-beta zeolite offer suitable redox sites. Meanwhile, the acid strength and distribution of H-beta zeolite are modulated by the interaction between VOx species and zeolite framework. The acid properties of the catalysts with different calcination temperatures are analyzed by pyridine-Fourier transform infrared spectra and NH3 temperature-programmed desorption measurements, and the selectivity to DMMx shows a linear relationship with the Bronsted/Lewis acid sites ratio, indicating that the catalysts with higher B/L ratio are effective for the DMMx formation. In addition, the NH3-TPD results confirm that more acid sites with strong strength for the catalysts are in favor of the C-O chain growth to synthesize larger DMMx molecules. These results above are helpful for us to well understand the active sites and design the effective catalysts to synthesize DMMx with longer C-O chain.