This study examines the impact of bio-organic fertilizers on the physiological characteristics and leaf nutrient content of “Qi-Nan” agarwood seedlings. The aim is to provide a theoretical foundation for the efficient cultivation of “Qi-Nan”. The experiment was conducted under field conditions using a Randomized complete block design. The control group received no fertilization, while the three fertilizer-treated groups received vermicompost, sheep manure, or microbial organic fertilizer. The results indicate that the application of bio-organic fertilizer significantly promoted the growth of plant height and diameter, increased the contents of soluble proteins, soluble sugar, chlorophyll, and leaf nutrients; and decreased the contents of free proline, peroxidase, malondialdehyde, and other stress indices. Redundancy analysis showed that the main factors affecting the seedlings height and diameter were free proline, chlorophyll, total nitrogen, and total potassium. Principal component analysis and membership function analysis revealed significant variations in growth, physiological characteristics, and nutrient content among the three fertilizer groups and the control group, with vermicompost > microbial organic fertilizer > sheep manure > no fertilization. In conclusion, biological organic fertilizer significantly promotes the growth of “Qi-Nan” seedlings, and vermicompost is the most effective fertilizer in this experiment.