Abstract: Box-Behnken response surface methodology (BBD-RSM) was used to optimize the purification process of total flavonoids from Eucalyptus smith leaf (TFESL) by macroporous adsorption resin, and its antioxidant and nitrosation inhibition activities were investigated. The adsorption - desorption capacities of nine types of resins, namely D101, NKA-9, XAD-8, NKA-Ⅱ, AB-8, DM-130, X-5, H103, and DA201, were investigated for TFESL using static adsorption. XAD-8 macroporous adsorption resin was selected for the enrichment and purification of TFESL. Based on the results of dynamic adsorption experiments (considering factors such as sample concentration, sample volume, flow rate, and eluent concentration), and using the transfer rate of TFESL as an indicator, the BBD-RSM was selected to optimize the process conditions for purifying TFESL using XAD-8 macroporous adsorption resin. The scavenging effects of crude and purified TFESL on DPPH·, ·OH, ABTS⁺·, and O₂⁻·, as well as their nitrosation inhibition effects, were examined. The optimal purification conditions were as follows: Sample concentration of 3.50 mg/mL, sample volume of 1.4 BV, flow rate of 1.5 BV/h, and eluent concentration of 77.00%. This process increased the purity of TFESL from 9.93% to 54.83%. The scavenging of free radicals and inhibition of nitrosation by TFESL exhibited dose-dependent relationships. The scavenging rate of purified TFESL at a concentration of 10.00 mg/mL for DPPH· was 95.30% (IC₅₀ = 1.28 mg/mL), and the IC₅₀ values for the scavenging of nitrite and inhibition of nitrosamine synthesis by purified TFESL at 2.50 mg/mL were 2.08 mg/mL and 0.60 mg/mL, respectively. The purification method optimized by the BBD-RSM is stable and feasible, and can be used for the purification of TFESL. The purified TFESL exhibited strong antioxidant and nitrosation inhibitory activities, providing experimental data and reference for the further development and utilization of Eucalyptus smithiileaves.