Mitogen-activated protein kinase kinases (MAPKKs or MKKs) are essential components of the highly evolutionarily conserved mitogen-activated protein kinase (MAPK) cascade, which play crucial roles in response to a variety of biotic and abiotic stresses and immune responses. However, very little information is available about the MKKs in S. maximus. In order to detect the roles of the MKKs in response to biotic and abiotic stresses in Scophthalmus maximus, we first identified the MKKs of S. maximus at the whole genome level through bioinformatics methods, and then analyzed the expression patterns of MKKs in different tissues and under different biotic and abiotic stresses using multiple stress-related RNA-seq datasets. As a result, a total of 9 MKKs of S. maximus (SmMKKs) were identified, and they were unevenly distributed on 7 chromosomes. Physicochemical characteristics, secondary structure and subcellular localization of the proteins they encode were predicted, respectively. Phylogenetic analysis revealed that SmMKKs were classified into 5 subfamilies. Conserved motifs, intron-exon structure and multiple sequence alignment not only provided evidences for the classification of MKK subfamilies, but also revealed high levels of conservation in evolution within and between subfamilies. Expression patterns of SmMKKs in distinct tissues and under diverse abiotic and biotic stresses were examined using multiple published RNA-seq datasets. As a result, SmMKKs showed obviously tissue-specific expression. In addition, SmMKK6a was extremely significant differentially expressed after infection with both Enteromyxum scophthalmi and Megalocytivirus. After heat stress, SmMKK6a also showed extremely significant differential expression. Furthermore, SmMKK4a, SmMKK4b, SmMKK6a and SmMKK7 were extremely significant differentially expressed after high- or low- salinity stresses. Among these candidate stress-responsive MKK genes, SmMKK6a showed extremely significant response to both abiotic and biotic stresses, demonstrating its potential functions in comprehensive anti-stress. This study may be the first to systematically identify and analyze the MKK gene family in turbot. The results not only demonstrate that SmMKKs play crucial roles in response to various biotic and abiotic stresses, but also provide important theoretical support for the development of molecular selective breeding for comprehensive stress-resistance in S. maximus.