Despite rigorous theoretical and clinical training, nursing students face emotional and academic challenges that can harm their well-being. Nonetheless, there is a dearth of research investigating the impact of laughter yoga (LY) on nursing students' well-being, perceived stress, and self-efficacy. The aim of this single-blinded, two-armed randomized controlled trial is to evaluate the effect of LY on nursing students' well-being, perceived stress, and academic self-efficacy (ASE). A total of five sessions of LY were applied to the intervention group. The analysis included a total of 83 nursing students (intervention group n = 41 and control group n = 42). Repeated-measures multivariate analysis of variance (MANOVA) and paired-samples t-test were used for data analysis. Results revealed that the intervention group showed a significant increase in well-being (p < .001) and a decrease in perceived stress after LY (p < .01). However, statistically significant differences between the intervention and control groups were not found in well-being, perceived stress, and ASE (p > .05). These results indicate the potential effects of LY on the students' well-being and perceived stress. Community mental health nurses and educators can use LY to improve well-being in universities.