Introduction Dupilumab has recently been approved for the treatment of moderate to severe atopic dermatitis (AD) in adults. Daily practice data on dupilumab treatment are scarce. Objective To study the effect of 16‐week treatment with dupilumab on clinical response and serum biomarkers in adult patients with moderate‐severe AD in daily practice. Methods Data were extracted from the BioDay registry, a prospective multicenter registry. Sixteen‐week clinical effectiveness of dupilumab was expressed as number of patients achieving EASI‐50 (Eczema Area and Severity Index) or EASI‐75, as well as patient‐reported outcomes measures (Patient‐Oriented Eczema Measure, Dermatology Life Quality Index, Numeric Rating Scale pruritus). Twenty‐one biomarkers were measured in patients treated with dupilumab without concomitant use of oral immunosuppressive drugs at five different time points (baseline, 4, 8, 12, and 16 weeks). Results In total, 138 patients treated with dupilumab in daily practice were included. This cohort consisted of patients with very difficult‐to‐treat AD, including 84 (61%) patients who failed treatment on ≥2 immunosuppressive drugs. At week 16, the mean percent change in EASI score was 73%. The EASI‐50 and EASI‐75 were achieved by 114 (86%) and 82 (62%) patients after 16 weeks of treatment. The most reported side effect was conjunctivitis, occurring in 47 (34%) patients. During dupilumab treatment, disease severity‐related serum biomarkers (TARC, PARC, periostin, and IL‐22), eotaxin‐1, and eotaxin‐3 significantly decreased. Conclusion Treatment with dupilumab significantly improved disease severity and decreased severity‐related serum biomarkers in patients with very difficult‐to‐treat AD in a daily practice setting. This study evaluated the clinical effectiveness and safety of 16‐weeks of dupilumab treatment in adults with AD. Dupilumab treatment significantly suppressed disease severity‐related serum biomarkers and eosinophil chemokines. By the end of the treatment, the EASI‐50 and EASI‐75 was achieved by 86% and 62% of patients, respectively. Abbreviations: AD: Atopic dermatitis; DLQI: Dermatology life quality index; EASI: Eczema area and severity index; IGA: Investigators global assessment; NRS: Numeric rating scale; PARC: Pulmonary and activation‐regulated chemokine; POEM: Patient‐oriented eczema measure; TARC: Thymus‐ and activation‐regulated chemokine