As a candidate anode material for Li-ion batteries, Bi-based materials have attracted extensive attention from researchers due to their high specific capacity, environmental friendliness, and simple synthesis methods. However, Bi-based anode materials are prone to causing large volume changes during charging and discharging processes, and the effect of these changes on lithium storage performance is still unclear. This work introduces that Bi/C nanocomposites are prepared by the Bi-based MOF precursor calcination method, and that the Bi/C nanocomposite maintains a high specific capacity (931.6 mAh g−1) with good multiplicative performance after 100 cycles at a current density of 100 mA g−1. The structural evolution of Bi/C anode material during the first cycle of charging and discharging is investigated using in situ synchrotron radiation SAXS. The SAXS results indicate that the multistage scatterers of Bi/C composite, used as an anode material during the first lithiation, can be classified into mesopores, interspaces, and Bi nanoparticles. The different nanostructure evolutions of three types of Bi nanoparticles were observed. It is believed that this result will help to further understand the complex reaction mechanism of Bi-based anode materials in Li-ion batteries.