Start tuning your guitar by plucking the 6th string. It is the first string you see as you look down on your guitar; this string is also known as the low E string. When you pluck the string, the tuner shows if you need to tune the string up or down. Pluck the string and adjust the peg as needed.
The string is in tune when the "needle" hovers in the middle of the tuner window. Repeat for each string on your guitar; that's it, your guitar is in tune.
You can access the tuner from the pause menu of a song (both Practice and Play mode) and from the main game play screen.
When you access the tuner, the Auto mode is toggled green. This means that when you pluck a string, the tuner suggests which string you are tuning. Tuning in manual mode can be useful if the strings are too out of tune, or if you are tuning new strings for the first time.
To start manual tuning, select a string on the tuner screen. When you do this, you hear what the tuned string should sound like. Pluck the string on your guitar and adjust the peg as needed. Repeat this with each string to tune them.
With the Audio setup button, you can access the Advanced audio settings in the Game tab.
Watch this video from the Yousician YouTube channel for a more in-depth tutorial about tuning your guitar.
If Yousician can't hear you at all, check troubleshooting tips here.