In this paper, trace lead in marine biological samples was determined by using flow injection ( FI ) air mixture adsorption preconcentration and twice gas isolation elution method in knotted reactor (KR) coupled with flame atomic absorption spectrometry (FAAS) . Air, complexing reagent and lead solution were mixed on-line during the preconcentration step, and the introduction of air greatly improved the adsorption effects of the lead chelate on the inner walls of KR. By the introduction of air flow before elution and adding part of air flow during elution, through these two parts of the introduction of air flow bubble partition, the dispersion of the lead chelate in KR was greatly reduced, and the peak of the atomic absorption signal was increased, and the enhancement factor (Enhancement factor, EF) was increased. By the combination with the KR of the air mixing sorption preconcentration and twice gas isolation elution method, the KR adsorption effect and elution effect reached a higher level, and compared with high back pressure and short lifetime of the micro column, KR has the characteristics such as acid and alkali resistance, low back pressure and almost unlimited lifetime. What’s more, it has no critical requirement for the peristaltic pump and is not easy to cause leakage of the solution. In the elution process, 1 s air flow was used as a spacer at the fifth second, instead of entering the air flow after the entire elution process was completed. The advantage of this method is not only being eluted completely, but also greatly reducing the dispersion of the analyte during the elution process, and the value of the absorption signal is greatly increased. If the elution time is simply put down to 5 s, then the elution will be very incomplete, the tube being not clean, a large number of analytes still remaining in the tube, and the remnants of the analyte were brought into the analysis of the next sample. All the experimental data will be inaccurate, and the value of data utilization will be lost. So complete elution is necessary and must ensure a certain elution time. As for the elution process, the elution of the analyte decreased gradually, in the past elution method, until the entire process is performed after elution, the analyte in the elution dispersion has been quite serious, which leads to the great reduction of absorption signal value. Operational parameters such as types, concentration and pH of complexing agents, sample preconcentration time and flow rate, flow rate and time of air mixing sorption process, the first ventilation time, pre elution time, the second ventilation time were optimized for the purpose of determining trace lead. Using the optimized conditions, a linear relationship existed between the peak absorbance and the concentrations of lead in the range of 0.005～0.6 mg·L-1 respectively. The method was applied in the determination of trace lead in marine biological samples with a detection limit(3σ) of 2.2 μg·L-1. In this paper, the detection results of FAAS method, traditional FI-KR-FAAS air mixing sorption preconcentration method and normal elution method were compared with this method, and it showed that the new method obtained the highest enhancement factor value. This method was applied to detect the trace lead in biological samples such as eel, pawn, slipper lobster, sturgeon, tonguefish and mussel. The trace lead content in these biological samples were among 0.34~1.92 μg·g-1. When the 1.0 μg·g-1 standard Lead solution was added, the recovery was 93.5%~96.4%, and the relative standard deviation (RSD) was 0.52%~2.94%. The method of FI-KR-FAAS twice gas isolation elution for the determination of trace lead in marine biological samples obtains several merits. It has good preconcentration effect, high precision, high degree of accuracy and high enhancement factor. This method can satisfy the analysis requirement for the marine biological samples.