Cotton stalk biochar (CSCs) as anode material of lithium-ion battery (LIB) is studied. The effect of particle size on structural and electrochemical characteristics of derived carbon materials were investigated. There was a significant change on the structural characteristics of pyrolytic carbon when the cotton stalk particle size was less than 200 μm, especially the pore below 4 nm. The carbonized products obtained from cotton stalk with particle size of 100–200 and 450–2000 μm reached the maximal interlayer spacing (0.388 nm) and pore volume (0.285 cm3g-1). Meanwhile, the better tubular structure existed in the pyrolytic carbon produced from cotton stalk with larger particle size. The biochar prepared from cotton stalk with the particle size of 450–2000 μm exhibited the largest Li+ diffusion coefficient (1.47 × 10−11 cm2 s−1), and the best electrochemical performance, which showed a larger specific capacity of 271.7 mAhg−1 after 100 cycles at 0.1 Ag-1. The structure of biochar can be effectively controlled through changing the particle size of cotton stalk, which provides a more low-energy and environmentally friendly idea for the preparation carbon materials in the future.