It is very important to investigate antimony geochemical behavior in order to identify its source, or reveal contamination processes, since antimony and its compounds are considered to be pollutants of high priority by the Environmental Protection Agency of the United States (USEPA). However, the concentration of antimony in most geological samples is very low, and its stable isotope mass difference is only 1.6%. An antimony preconcentration method for isotope analysis using modified Thiol Cotton Fiber has been developed. Using this new method, the recovery of antimony was 99.5 ± 3.6% (n = 3) and blank values were <0.1 ng. The standard error of the ε(123)Sb isotope ratios in the proposed method is only 0.4ε, much smaller than those of the previous method. The proposed method is therefore effective in the preconcentration and separation of antimony for antimony isotope analysis.