In patients with relapsing remitting multiple sclerosis (RRMS), the effect of interferon-beta (INFb) on health-related quality of life (HR-QoL) is not firmly documented. The objective of this study is to assess HR-QoL during 2 years of treatment with intramuscular INFb and its correlation with disability. In 36 neurological practices in the Netherlands (17), Belgium (16), United Kingdom (2) and Luxemburg (1), 284 RRMS patients were treated with intramuscular INFb-1a. Physical and mental domains of HR-QoL were measured by the MS54 Quality of Life (MS54QoL) questionnaire, and disability was assessed by the Multiple Sclerosis Functional Composite (MSFC) (Timed 25-Foot Walk Test [Timed 25-FWT], 9 Hole Peg Test [9-HPT], Paced Auditory Serial Addition Test [PASAT]) at baseline and at months 3, 6, 12, 18 and 24. Expanded Disability Status Scale (EDSS) score was assessed at baseline and month 24. Pearson's correlation coefficients were determined and predefined factors were analyzed for relation to HR-QoL after baseline by stepwise regression analyses on physical and mental scores. 204 patients (71.8%) completed 2 years of treatment. Mean values for MS54QoL increased from 56.6 to 61.0 for physical (p < 0.05) and from 57.2 to 61.1 for mental domain (p = 0.07). Correlations between physical domain and MSFC was -0.40 (p < 0.05), and between mental domain and MSFC -0.24 (p < 0.05). MSFC and EDSS did not change. Increase of physical MS54QoL was associated with lower age, lower EDSS, less time for Timed 25-FWT, and higher PASAT score at baseline. Increase of mental MS54QoL was associated with higher PASAT and lower EDSS. Patients who discontinued INFb had lower physical or mental HR-QoL at baseline. In RRMS patients, 2 years of treatment with intramuscular INFb-1a is associated with an increase in HR-QoL, especially in younger patients with low disability.