Indicators of health have shown improvements in the Swedish working population during the past decades, but with the exception of low-skilled women. Earlier research has shown that assistant nurses belong to an occupational group, which has the lowest share of individuals with long-term health in the total Swedish workforce. Sick leave research has mostly focused on determining risk factors for the development of diseases and dysfunctions. In the process of acquiring knowledge about mechanisms for sick leave, it has become obvious that there is also a need to focus on what contributes to work ability. The aim in this study was to explore what promotes sustainable work ability in female assistant nurses working in care of older people. Associations between factors related to work, health, lifestyle and sustainable work ability were investigated in a cross-sectional nested case-control study in a cohort of 366 female assistant nurses. Data were collected in self-reports in a questionnaire. Odds ratios were calculated, and a multiple logistic regression analysis was performed with sustainable work ability as the dependent variable. Sustainable work ability was associated with good self-rated health, a BMI <30, ability to sleep well, recuperation, low stress level, support from family and friends, being over 25 years of age at birth of the first child, and control over one's own life in the ≥50 age group. In the logistic regression analysis, the significant associations were good self-rated health, being over 25 years of age at the birth of the first child and recuperation. In conclusion, the study showed that self-rated health and factors in private life are important for assistant nurse's sustainable work ability over time. The results from this study could be applied in health promotion work for employees in the care sector to strengthen and enhance sustainable work ability.