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.