At the beginning of the scene, Romeo and Juliet have spent the night together, and Romeo must leave before sunrise to avoid detection by the Capulet family.
As Romeo remarks upon the approach of morning after hearing the lark, Juliet believes he is referencing the nightingale, a bird typically associated with passion, love, and melancholy. She says that the sound is in truth the nightingale, not the lark, and tries to convince Romeo to stay.
However, Romeo insists it is indeed the lark, symbolizing the arrival of day, and he must go. His reference to the lark underscores the reality of the situation and the need to part ways.
The song of the lark signals the end of the night and serves as a reminder of the harsh reality that threatens the love between Romeo and Juliet. It symbolizes the constraints and limitations imposed upon their romance, foreshadowing the tragic events that will befall them.