Objective To evaluate the effectiveness of the portfolio-based formative assessment in bilingual teaching of pediatric nursing. Methods Quasi-experimental design was adopted. A total of 97 bilingual experimental class students (four-year program) from grade 2013 and grade 2014 who received Pediatric Nursing education from February 2016 to July 2017 in Guangzhou Medical University were included. Fifty-eight students from grade 2014 were the experimental group with the new formative assessment based on portfolio, while thirty-nine students from grade 2013 were the control group with traditional assessment. The final theory examination scores, the self-directed learning ability scale and the academic self-efficacy scale were compared between two groups. Results The score of the English questions in the final theory examination of experimental group was higher than that of control group[(21.95±2.92) vs. (20.59±2.52)]; the total score of self-directed learning capacity and the total score of academic self-efficacy in experimental group were significantly higher than those in the control group [(73.76±7.61) vs.(69.87±9.68), (79.47±7.97)vs.(75.92±8.34); all P<0.05]. Conclusions Formative assessment based on portfolio improves English performance, the self-directed learning capacity and academic self-efficacy.