Telomere length (TL) is a promising biomarker for age-associated diseases and cancer. Single-molecule studies of human TL are advancing rapidly, providing unprecedented insights into the dynamics and variability of telomeres at the single-molecule level. TL is commonly reported as average TL or relative TL, depending on the methods used. However, average TL, short TL, and long TL have distinct significances: average TL serves as a general biomarker for aging, short TL indicates the risk of age-related diseases, and long TL is associated with certain cancers and all-cancer mortality. Thus, the TL distribution is more important than the average TL alone. Single-molecule techniques measure TL one telomere at a time, offering quantitative TL distributions that are crucial for understanding telomere biology. In this review, we focus on various TL measurement techniques, with particular emphasis on single-molecule methods. Single-molecule studies of human TL are poised to play a pivotal role in advancing our understanding and clinical management of age-associated diseases and cancer.