Choline receptor blocker succinylcholine (suxamethonium [SUX]) is a depolarizing neuromuscular blocking agent to relax muscles. It is often used in clinic to conduct auxiliary endotracheal intubation as a surgical anesthetic inducer, which can cause respiratory depression and cardiac arrest. In recent years, SUX was occasionally used for theft and crime by daubing on darts, resulting in intoxication or even death. Regarding the rapid detection demand of SUX in first aid and emergency response, a bit of challenges still existed, especially towards biomedical samples due to its properties of high hydrophilic, alkali‐liable, low onset concentration, and susceptibility to cholinesterase. In this report, we described an on‐site surface‐enhanced Raman spectroscopy (SERS) method on SUX in the first time. We found that key parameters facilitated this SERS method including MgI2‐induced appropriate aggregation and higher pH, in which enhanced electrostatic interaction between analyte and AuNPs occurred, and larger adsorption capacity of AuNPs, which makes the sensitive measurement of alkali‐liable SUX become possible. Coupled with simple and optimized pretreatment procedures, successful measurement of SUX in plasma and urine samples was also achieved. The limit of detection reached 1 μg/L in plasma and 10 μg/L in urine. We regarded acids for precipitating abundant proteins and inhibited cholinesterase activity in plasma and proteins for assisting antiagglomeration of AuNPs when coexisted with plenty of inorganic salts in urine, as the critical parameters in sample pretreatment. We hope this method can find its great potential in forensic and clinical diagnosis in case of SUX poisoning or intoxication.