The influence of rolling reduction and temperatures at room and liquid nitrogen on the formation of deformation-induced martensite and microstructural evolution in a β-ZrTiAlV alloy was investigated by X-ray diffraction (XRD), optical microscopy (OM) and electron backscatter diffraction (EBSD) technique. The analysis of the phase composition and microstructure of the rolled alloy indicated that a deformation-induced martensite phase transformation process from body-centered cubic β to closed-packed hexagonal α＇accompanying with (102) <110> twinning in the α＇phase were simultaneously detected. With the increase of rolling reduction, deformation-induced phase transformation and the volume fraction of martensite variant were promoted. Nevertheless, lower temperature (liquid nitrogen) resulted in the inhibition of α＇martensite transformation and the promotion of the (102) <110> twinning. When the specimen was subjected to the rolling reduction of 5%, plate-like deformation-induced martensite started appearing in the equiaxed parent β grains. However, when the rolling reduction increased to 10%, the deformation-induced martensite would be generated fully within some β grains.