Abstract: Gold, as one of the important elements in critical minerals, high-accuracy and high-precision gold analytical testing methods are of great significance for revealing the genesis of geological minerals and mineral resource exploration. Although activated carbon adsorption-flame atomic absorption spectrometry (FAAS) has been widely used for the determination of gold in rock and mineral samples, the quality of activated carbon exerts a significant impact on the analytical results of gold. Conventional activated carbon is characterized by high ash content and low adsorption rate (only 90%-92%), which limits its application efficiency. Based on the high efficiency and environmental friendliness of spent mushroom substrate (SMS) biochar, this study established an analytical method for gold determination using SMS biochar adsorption coupled with FAAS. Single-factor experiments confirmed that carbonization temperature, ultrasonic time, modifier concentration, and adsorption time significantly affect the gold adsorption efficiency of SMS biochar. By optimizing the preparation conditions via response surface methodology (RSM), the optimal preparation conditions were determined as follows: carbonization temperature of 505℃, ultrasonic time of 54min, modifier concentration of 9%, and adsorption time of 170min. Under these conditions, the gold adsorption rate reached over 98%. Using SMS biochar prepared under the optimized conditions as the adsorbent, spiked recovery tests were carried out on 4 gold ore certified reference materials (CRMs) with different gold contents and 4 actual samples by FAAS. For the gold CRMs, the relative error (RE) between the determined values and the certified values ranged from 0.05% to 1.59%, and the relative standard deviation (RSD, n=6) was in the range of 0.30%-3.41%. For the actual samples, the spiked recovery rate of gold varied from 96.8% to 101.4%. The accuracy, precision, and spiked recovery rate all meet the quality requirements for the analysis of gold chemical components in rock and mineral samples specified in the Specification for Quality Management of Geological and Mineral Laboratory Testing (DZ/T 0130.3—2006). This method is suitable for the adsorption and analysis of gold in gold ores and has the potential for popularization and application in the field of gold determination in rocks and minerals.