Studying the relationship between agricultural irrigation water requirements (IWR) and water supply is significant for optimizing the sustainable management of water resources in Tarim River Basin (TRB). However, the related studies have not quantified the total IWR and the imbalance of irrigation water supply and requirements in the TRB. The study analyzed the spatial-temporal variations of IWR by a modified Penman–Monteith (PM) method during 1990–2015. Five major crops—rice, wheat, maize, cotton, and fruit trees—are chosen for calculating the IWR. It was found that the IWR increased significantly, from 193.14 × 108 m3 in 1990 to 471.89 × 108 m3 in 2015, for a total increase of 278.74 × 108 m3. For the first period (1990–2002), the total IWR remained stable at 200 × 108 m3 but started to increase from 2003 onwards. Significantly more irrigation water was consumed in the oasis regions of the Tienshan Mountains (southern slope) and the Yarkand River (plains). Furthermore, there was an intensified conflict between IWR and water supply in the major sub-basins. The ratios of IWR to river discharge (IWR/Q) for the Weigan-Kuqa River Basin (WKRB), Aksu River Basin (ARB), Kaxgar River Basin (KGRB), and Yarkand River Basin (YRB) were 0.93, 0.68, 1.05, and 0.79, respectively. The IWR/Q experienced serious annual imbalances, as high flows occurred in July and August, whereas critical high IWR occurred in May and June. Seasonal water shortages further aggravate the water stress in the arid region.