The reason I asked for the brand/model of the guitar is some are easier to tune than others, depending on the quality. Also, depending on the age of the guitar, the tuning pegs may be worn out, particularly on that one string. Furthermore, if the strings are very old and worn they may very likely not tune easily or stay in tune well. Lastly, having used many computerized guitar programs over the years, nothing beats a real tuner. You should consider going out and buying one.