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Reliable serum biomarkers are of immense need for diagnostic purposes of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS)—a disabling and complex disease for which diagnosis is mainly based on clinical symptoms. The aim of this study was to evaluate a possible diagnostic potential of activin B by directly comparing 134 cases of ME/CFS with 54 healthy controls. Analyses of human activin B level in plasma samples were performed using a validated human activin B ELISA assay. The results of the study show that activin B levels did not differ statistically significantly between ME/CFS patients and healthy controls (<i>p</i> = 0.6511). No gender or age-related differences in activin B levels were observed in the ME/CFS group and healthy controls. The level of activin B tended to decrease with increasing visual analogue scale score (<i>r</i> = −0.2004; <i>p</i> = 0.5085) nevertheless the results obtained so far does not support the clinical utility of activin B as a biomarker for ME/CFS.