Currently, due to the special functions and potential application values, rare sugars become the hot topic in carbohydrate fields. l-Ribulose, an isomer of l-ribose, is an expensive rare ketopentose. As an important precursor for other rare sugars and l-nucleoside analogue synthesis, l-ribulose attracts more and more attention in recent days. Compared with complicated chemical synthesis, the bioconversion method becomes a good alternative approach to l-ribulose production. Generally, the bioconversion of l-ribulose was linked with ribitol, l-arabinose, l-ribose, l-xylulose, and l-arabitol. Herein, an overview of recent advances in the metabolic pathway, chemical synthesis, bioproduction of l-ribulose, and the potential application of l-ribulose is reviewed in detail in this paper.