With the strict regulation of brominated flame retardants, organophosphate esters (OPEs) have been extensively used as replacements. Increasing concerns on OPEs have aroused due to their extensive distribution in the environment and humans, as well as their potential toxicities. Recent studies have demonstrated that some organophosphate di-esters are even more toxic than their respective tri-esters. This review summarized the current state of knowledge on the analytical methodologies (including sample collection and preparation, instrumental analysis, and the feasibility of each potential human matrix), as well as the occurrences of OPEs and/or their metabolites (m-OPEs) in various human matrices. Organophosphate esters are readily metabolized in human thus only limited studies reported their occurrences in blood and breast milk, whereas abundant studies are available regarding the occurrences of m-OPEs rather than OPEs in urine. Since none of the matrix is suitable all the time, appropriate matrix should be selected depending on the aims of biomonitoring studies, e.g., high throughput screening or body burden estimation. Biomonitoring with non-invasive matrices such as hair and/or nail is useful to screen specific populations that might be under high exposure risks while urine is more suitable to provide valuable information on body burden. In terms of urinary monitoring, specific biomarkers have been identified for some OPE compounds, including tri(2-butoxyethyl) phosphate, tri(1,3-dichloro-2-propyl) phosphate, tri(2-chloroethyl) phosphate and tri(1-chloro-2-propyl) phosphate. Further studies are required to identify suitable urinary biomarkers for other OPE compounds, especially the emerging ones.