To investigate the relationship between Life's Essential 8 (LE8) and Phenotypic Age Acceleration (PhenoAgeAccel) in United States adults and to explore the impact of LE8 on phenotypic biological aging, thereby providing references for public health policies and health education. Utilizing data from the National Health and Nutrition Examination Survey (NHANES) conducted between 2007 and 2010, this cross-sectional study analyzed 7,339 adults aged 20 and above. Comprehensive assessments of LE8, PhenoAgeAccel, and research covariates were achieved through the integration of Demographics Data, Dietary Data, Laboratory Data, and Questionnaire Data derived from NHANES. Weighted generalized linear regression models and restricted cubic spline plots were employed to analyze the linear and non-linear associations between LE8 and PhenoAgeAccel, along with gender subgroup analysis and interaction effect testing. (1) Dividing the 2007-2010 NHANES cohort into quartiles based on LE8 unveiled significant disparities in age, gender, race, body mass index, education level, marital status, poverty-income ratio, smoking and drinking statuses, diabetes, hypertension, hyperlipidemia, phenotypic age, PhenoAgeAccel, and various biological markers (p < 0.05). Mean cell volume demonstrated no intergroup differences (p > 0.05). (2) The generalized linear regression weighted models revealed a more pronounced negative correlation between higher quartiles of LE8 (Q2, Q3, and Q4) and PhenoAgeAccel compared to the lowest LE8 quartile in both crude and fully adjusted models (p < 0.05). This trend was statistically significant (p < 0.001) in the full adjustment model. Gender subgroup analysis within the fully adjusted models exhibited a significant negative relationship between LE8 and PhenoAgeAccel in both male and female participants, with trend tests demonstrating significant results (p < 0.001 for males and p = 0.001 for females). (3) Restricted cubic spline (RCS) plots elucidated no significant non-linear trends between LE8 and PhenoAgeAccel overall and in gender subgroups (p for non-linear > 0.05). (4) Interaction effect tests denoted no interaction effects between the studied stratified variables such as age, gender, race, education level, and marital status on the relationship between LE8 and PhenoAgeAccel (p for interaction > 0.05). However, body mass index and diabetes manifested interaction effects (p for interaction < 0.05), suggesting that the influence of LE8 on PhenoAgeAccel might vary depending on an individual's BMI and diabetes status. This study, based on NHANES data from 2007-2010, has revealed a significant negative correlation between LE8 and PhenoAgeAccel, emphasizing the importance of maintaining a healthy lifestyle in slowing down the biological aging process. Despite the limitations posed by the study's design and geographical constraints, these findings provide a scientific basis for the development of public health policies focused on healthy lifestyle practices. Future research should further investigate the causal mechanisms underlying the relationship between LE8 and PhenoAgeAccel and consider cross-cultural comparisons to enhance our understanding of healthy aging.