Abstract: Gold, as an important element in critical minerals, requires high-accuracy and high-precision analytical methods, crucial for revealing the genesis of geological minerals and for 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 significantly impacts the analytical results for 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, an analytical method for gold determination using SMS biochar adsorption coupled with FAAS has been established. Single-factor experiments confirmed that carbonization temperature, ultrasonic time, modifier concentration, and adsorption time significantly affect the adsorption efficiency of SMS biochar for gold. 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 54 min, modifier concentration of 9%, and adsorption time of 170 min. 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 real 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 real samples, the spiked recovery rate of gold varied from 96.8% to 101.9%. The accuracy, precision, and spiked recovery rate all met the quality requirements specified in The Specification of Testing Quality Management for Geological Laboratories (DZ/T 0130.3-2006) for the analysis of gold in rock and mineral samples. 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. The BRIEF REPORT is available for this paper at http://www.ykcs.ac.cn/en/article/doi/10.15898/j.ykcs.202503180049.