The invention relates to a culture solution for preparing pancreatic beta cells by inducing directional differentiation of pluripotent stem cells, the culture solution comprises an AdvancedDMEM / F12 culture medium, glucose, sodium bicarbonate, human serum albumin (HSA), N2, B27, glutamine, vitamin C, a JNK inhibitor, a hedgehog pathway antagonist, EGF signal factors, FGF signal factors, a TGF-beta inhibitor, a WNT signal pathway activator, a Notch signal pathway inhibitor, a small molecule compound and trace elements, the ratios of the culture solution in different stages of cell culture are different, all the components play roles directionally in stages, and the pluripotent stem cells can be efficiently induced to be directionally differentiated into the pancreatic beta cells.