Vitamin D plays an important role in decreasing the risk of various musculoskeletal diseases and chronic illnesses by participating in the calcium, phosphate absorption, bone metabolism and many other metabolic pathways. Vitamin D deficiency among populations of different ages is common throughout the world, even in the developed countries. Accurate vitamin D analysis is crucial for quality control in pharmaceuticals and foods and assessment of human vitamin D nutritional status. This review briefly introduces the existing forms in different matrices, synthesis and metabolism process in humans of vitamin D, as well as the related diseases caused by its deficiency. The emphases are focused on the recent advances of sample pretreatment and analytical techniques of vitamin D and its analogues in pharmaceuticals, foods and biological samples in the last five years.