Abstract: To address the challenges of efficient refractory coal separation under the “Dual Carbon” policy and the safety and environmental shortcomings of traditional flotation reagents (kerosene and petrochemical diesel), this study evaluates the suitability and separation performance of safe, eco-friendly, and biodegradable bio-flotation reagents for specific refractory coal. Taking the 105^# refractory coal from the Zhengli Mine Coal Preparation Plant in Lanxian as the research object, the particle size distribution was first analyzed through fractional screening, with a focus on high-ash fine slime distribution. Flotation rate and step-wise release tests were used to evaluate flotability and identify suitable processes. Inverse Gas Chromatography (IGC) was employed to determine surface energy parameters, such as dispersive surface energy and acid-base surface energy, alongside the hydrophilicity index. Additionally, a collector index was introduced to compare the performance of the bio-collector against kerosene, dodecane, and hexadecane.A two-stage flotation process (“one roughing + one cleaning”) was then implemented, using clean coal yield, ash content, and tailing ash content as core indicators to optimize the dosage of the bio-flotation reagents (comprised of a bio-collector and bio-frother) and compare them with onsite reagents. The results indicate that the 105^# coal sample is significantly hydrophilic (index: 0.16), with high-ash fine slime (< 0.03mm) accounting for 43.70%, classifying it as difficult-to-float. The collector index follows the sequence: bio-collector (0.85) > hexadecane (0.81) > dodecane (0.77) > kerosene (0.76), demonstrating the superior collecting ability of the bio-reagent. The two-stage process successfully meets onsite production requirements; at an optimal dosage of 0.25 kg/t, clean coal ash is 12.63% and tailing ash is 63.67%. Compared to onsite reagents at equivalent dosages, the bio-reagent increases clean coal yield by 6.76 percentage points and tailing ash by 13.33 percentage points. At a constant yield of approximately 45%, bio-reagent consumption is reduced by 6.4%. At the same dosage of 0.3 kg/t, the bio-flotation agent increased the tailings ash content by 10.3 percentage points.Bio-flotation reagents not only resolve the safety and environmental issues of traditional reagents but also improve separation efficiency and tailing quality while reducing chemical consumption, aligning with the strategic needs for clean coal processing under the “Dual Carbon” framework.