Recently, gel chromatography has been demonstrated as an effective method for the separation of single-walled carbon nanotubes (SWCNTs) according to their electronic type and structure. The separation of SWCNTs was thought to result from the different affinity forces between the gel and various SWCNTs. Based on this method, we investigated the effect of ultrasonic time on the dispersion and separation of metallic and semiconducting SWCNTs. At a low ultrasonic power, with the increase of ultrasonic time, better monodispersed SWCNTs in sodium dodecylsulfate (SDS) aqueous solution were obtained. The UV-visible-near infrared (UV-Vis-NIR) absorption, Raman and photoluminescence (PL) spectroscopic characterizations confirmed that under the condition of ultrasonication (2 h), higher-purity metallic tubes and semiconducting tubes with different diameter distributions could be obtained. We believe that the control of the ultrasonication time may tune the mono-dispersity and the length of SWCNTs, which would further influence the difference in affinity forces between various SWCNTs and the gel, therefore leading to different separation results.