Strigolactones (SLs) are novel plant hormones that play crucial roles in regulating plant growth and development. The stereoisomers of SLs often exhibit distinct biological activities, making their chiral separation and analysis essential for understanding their regulatory roles in plant branching. This study focused on the chiral separation and characterization of SL stereoisomers, including GR24 (synthetic) and strigol (natural). A magnetic nanocomposite (Ni₃(HITP)₂@Fe₃O₄@rGO) was synthesized for SL extraction, combined with SFC-MS/MS for precise separation and quantification. The method exhibited excellent linearity (0.2-50 ng/mL), minimal matrix effects (1.64-2.17 %), and low detection limits (1.93-3.76 pg/mL). Recoveries ranged from 86.90 % to 106.37 % with RSDs of 2.56 %-13.54 %. Under nitrogen and phosphorus stress, only (+)-strigol was detected in the roots of Oryza sativa L., suggesting its regulatory role under stress conditions. This reliable method provides robust support for exploring the physiological roles of SL stereoisomers in plants, shedding light on their functions in stress response and development.