Abstract: We report an efficient method for the determination of iodide (I–) ions by using gold–iodide hybrid cluster ions on gold nanoparticles (Au NPs) modified mixed cellulose ester membrane (Au NPs-MCEM) by pulsed laser desorption/ionization mass spectrometry (LDI-MS). When I– ions were deposited and concentrated on the surfaces of Au NPs (32 nm) via strong Au+–I– interaction on the MECM, the Au NPs-MCEM was observed to function as an efficient surface-assisted LDI substrate with very low background noise. When pulsed laser radiation (355 nm) was applied, I– binding to Au NPs ions induced the enhancement of the desorption and ionization efficiency of gold–iodide hybrid cluster ions from the Au NPs surfaces. The reproducibility of the probe for both shot-to-shot and sample-to-sample (both less than 10%) ion production was also improved by the homogeneous nature of the substrate surface. Thus, it allows the accurate and precise quantification of I– ions in high-salinity real samples (i.e., edible salt samples and urine) at the nanomolar range. This novel LDI-MS approach provides a simple route for the high-speed analysis of I– ions with high sensitivity and selectivity in real biological samples.