Reliable, fast and cost efficient mercury analysis is paramount to assess Hg levels in a huge variety of matrices, from soil and water to a variety of foodstuffs. In this work, a novel concept was adopted for seafood and hair analysis, which combines a simplified methylmercury extraction and photochemical vapour generation of Hg0 with liquid chromatography and atomic fluorescence spectrometry (LC-AFS). This concept reduces the number of reagents required to methanol and APDC for extraction and separation of Hg species, and acetic acid, which when combined with UV generates volatile Hg0 in one single step. Here, we compare conventional chemical vapour generation (CVG) with photochemical vapour generation (PVG) for seafood and hair matrix. Our results show that the LC-PVG-AFS method offers lower detection limits and higher precision than that of LC-CVG-AFS measurements by a factor of two. The extraction and LC-PVG-AFS technique was validated using 7 seafood and hair reference materials, yielding methylmercury recoveries of 93.2% to 105%. Further validation was carried out by comparing results from methylmercury determination in 14 tuna samples by the LC-PVG-AFS method to results obtained by GC-AFS, showing R2 = 0.98 and a gradient of 1.0077, which indicates good correlation between the newly developed LC-PVG-AFS technique to an already validated GC-AFS method.