The smartphone is a widely used and rapidly growing phenomenon worldwide, and problematic smartphone use is common in our society. This study’s objective was to examine the gender difference of baseline and post-intervention skin conductance response (SCR) among smartphone users and explore the relationships among problematic smartphone use level, anxiety level, and SCR changes by evaluating SCR, the Zung Self-Rating Anxiety Scale score, and the Chinese version of the Smartphone Addiction Inventory (SPAI) score in a one-group baseline and post-test design. Sixty participants were recruited from two communities, and data were collected from April to June 2017. There was a significant difference in terms of SCR changes between young males and old males and between young females and old females. Additionally, the SCR changes in young females were significantly greater than those in young males with twofold mean difference. This study provides strong evidence supporting the effectiveness of SCR measurement for assessing problematic smartphone use (PSU) anxiety when users are in a withdrawal-like state. The SCR measurement can help healthcare providers identify cases with risk factors of PSU for early intervention.