Strong association has been observed between children blood Pb and Pb in house dust, suggesting the contribution of house dust ingestion to metal exposure to children. However, to accurately assess the health risk, there is a need to determine oral metal bioavailability in house dust. In addition, appropriateness of in vitro bioaccessibility assays as a surrogate of metal RBA has not been elucidated for house dust. Following method development, the bioavailability needs to be incorporated into risk assessment. For in vitro bioaccessibility method development, we collected house dust samples from uncontaminated areas across China and Pb/Zn mining areas, Hunan, China. In vivo mouse bioassays were applied to measure As, Pb, and Cd relative bioavailability (RBA), which were compared to bioaccessibility measured using in vitro assays including SBRC, IVG, DIN, PBET, and UBM to determine the most suitable in vitro method as a surrogate of animal bioassays. In addition, bioavailability/bioaccessibility measurement was coupled with the stable isotope ratio technique to identify dominant exposure pathway for children with elevated blood Pb living in Zhuhang, China, a town characterized with intensive wire rope production and residents living in mining/smelting districts, Hunan, China. For house dust from uncontaminated areas, Pb and As RBA was 29–60% and 22–87% based on in vivo mouse blood assay. When validated against in vivo RBA data, SBRC and DIN methods had the strongest potential to predict As and Pb bioavailability in the dust samples. However, for contaminated house dust, As and Pb RBA (7.64–37% and 7.21–52.1%) based on mouse liver and kidney assay was significantly lower, with Cd RBA being firstly reported to be 20.3–94.3%, suggesting the influences of contamination source on metal(loid) bioavailability. Among bioaccessibility methods, the single gastric phase of the UBM method could be used to predict bioavailability of co-contaminants in house dust from mining/smelting areas. For children in Zhuhang, when stable isotopic composition of children blood Pb was compared to that of total Pb in the environmental medias (house dust, soil, PM10, vegetables, rice, and drinking water), there was no match between blood Pb and Pb in any environmental media. However, when determined for bioaccessible Pb using the SBRC method, Pb isotopic composition in house dust showed a close agreement with blood Pb, suggesting that coupling stable isotope technique and bioavailable Pb analysis provided a more robust approach for identifying Pb exposure pathways than total Pb determinations. For residents living in mining/smelting site, As, Cd, and Pb exposure via house dust ingestion was compared to that via rice consumption based on RBA. Interestingly, for adults, rice was the main As contributor, while house dust ingestion was the major As contributor to children. However, for both adults and children, rice was the main source for Cd exposure, while house dust was the predominant Pb contributor. To confirm this, an overlap of stable Pb isotopic composition was observed between Pb in house dust and hair from the residents, while Pb signals in rice was different from hair Pb.