The extensive presence of anisakids in fish for human consumption has become a problem of food safety and quality. The aim of this study was to develop and assess the performance of a quantitative SYBR Green qPCR assay for the detection and quantification of Anisakis DNA in fish by-products. L3 nematode larvae of A. simplex (s.l.) (n = 510), A. physeteris (n = 3), Hysterothylacium sp. (n = 10) and Pseudoterranova sp. (n = 1), isolated from blue whiting, horse mackerel and monkfish, were used for the optimization of the molecular assay. In addition, molecularly typed larvae of A. simplex (s.s.) (n = 10) and A. pegreffii (n = 5) of the complex A. simplex (s.l.) were used for the specificity assay. Primers targeting the mitochondrial cytochrome c oxidase subunit II gene (COII) were selected. Analytical sensitivity and reproducibility were evaluated in a food matrix consisting of commercial fish-derived food spiked with larvae of A. simplex (s.l.). The assay proved to be specific for the three analyzed Anisakis species. A high reproducibility and sensitivity was detected, with a 95% limit of detection (LOD) of 0.30 ng (95%CI 0.15–1.50) of A. simplex (s.l.) DNA per gram of food matrix and an operative LOD of 1.50 ng after a PROBIT analysis. The assay was applied to study the presence of Anisakis in four types of processed commercial food, namely crab sticks, “gulas”, croquettes and burgers. Overall, 180 food samples from 15 commercial brands were studied, detecting Anisakis DNA in over half of them. The analyzed surimi-based products, “gulas” and crab sticks, showed the highest Anisakis burden (5.86 ± 0.69 and 4.68 ± 0.73 ng of Anisakis DNA per gram of food, respectively). Our results indicate that the optimized SYBR Green qPCR technique is an accurate and sensitive method that may improve detection of Anisakis in fresh and processed products.