BACKGROUND: Individuals with chronic diseases commonly engage in a sedentary lifestyle, which may exacerbate poor disease progression and increase the burden of care. Digital health interventions have been broadly used in promoting healthy lifestyles in recent decades, while their effectiveness on sedentary behavior (SB) remains inconsistent and inconclusive. OBJECTIVE: This review aimed to evaluate the effectiveness of digital health interventions in reducing SB among patients with chronic diseases. METHODS: PubMed, Embase, Scopus, Web of Science, CINAHL Complete, Cochrane Library, and ACM Digital Library were searched for randomized controlled trials published from January 2000 to October 2023. Two researchers independently screened studies and evaluated study quality. The revised Cochrane risk-of-bias tool was used to assess the risk of bias. Mean differences (MDs) were calculated for intervention effect comparison. RESULTS: Twenty-six trials were selected and 3800 participants were included. The mean age was 57.32 (SD 9.91) years. The typical chronic diseases reported in the studies included obesity (n=6), arthritis (n=5), coronary artery disease (n=4), cancer (n=4), type 2 diabetes mellitus (n=3), metabolic syndrome (n=2), and stroke (n=2). Phone, web, and activity trackers were 3 digital technologies adopted in the interventions and they were used in combination in most studies (18/26, 69.2%). The functions included facilitating self-monitoring of SB, reminding interruption of long undisturbed sitting, and promoting goal attainment. Approaches targeting SB reduction included standing (n=6), walking (n=9), light physical activity (n=5), moderate to vigorous physical activity (n=4), screen time limitation (n=2), and contextual-related activities based on patients' preference (n=4). The majority (80.8%) of studies had a low to moderate risk of bias. Meta-analysis revealed significant decreases in overall sitting time (MD -30.80; 95% CI -49.79 to-11.82; I2=65%; P=.001), pre-post sitting time changes (MD -50.28; 95% CI -92.99 to -7.57; I2=92%; P=.02), and SB proportions (MD -4.65%; 95% CI -7.02 to -2.28; I2=20%; P<.001) after digital health interventions, compared with nondigital interventions such as usual care, wait-list, or other active controls, with a small effect size (Cohen d=-0.27 to -0.47). No significant differences in the length of sedentary bouts and breaks were found. Subgroup analyses showed that studies with objective SB measurements and those younger than 65 years had significant reductions in sitting time. CONCLUSIONS: Digital health interventions significantly reduced the SB among patients with chronic illness. More research with rigorous design to promote a long-term decrease in sitting time, differentiate primary and compensatory SB reductions, and explore the underlying mechanisms is needed.