Deliberately, there is some distance between the separation line and the content of the lower box part. Adding the draft
options helps to understand what happens:
\documentclass[a4paper]{article}
\usepackage[scale=.85]{geometry}
\usepackage{tcolorbox}
\tcbuselibrary{skins,xparse}
\begin{document}
\begin{tcolorbox}[skin=bicolor,draft,title=Main Title,
colframe=red!75!black,colback=blue!10!white,colbacklower=green!10,width=0.5\linewidth,height=0.5\linewidth]
The upper part. \par ~~~\par ~~~\par ~~~\par ~~~\par ~~~\par ~~~\par ~~~\par ~~~\par ~~~
\tcblower
\tcbsubtitle[]%
{My subtitle}
The lower part.
\end{tcolorbox}
\end{document}

The distance between the bicolor
separation line and the content of the lower part is middle
(default: 2mm) plus boxsep
(default: 1mm). You see these distances in white and pink color.
The subtitle starts exactly at the begin of the lower part (you may spot the dashed line at the top of the subtitle).
To move the subtitle upwards to the separation line, one could use enlarge top initially by
to change the bounding box of the subtitle. This works as long as the height of the subtitle is larger than the corrected distance. Actually, I would add a small additional distance to avoid optical glitches when two lines are matched, e.g. use 3.1mm
for the default values:
\documentclass[a4paper]{article}
\usepackage[scale=.85]{geometry}
\usepackage{tcolorbox}
\tcbuselibrary{skins,xparse}
\begin{document}
\begin{tcolorbox}[skin=bicolor,title=Main Title,
colframe=red!75!black,colback=blue!10!white,colbacklower=green!10,width=0.5\linewidth,height=0.5\linewidth]
The upper part. \par ~~~\par ~~~\par ~~~\par ~~~\par ~~~\par ~~~\par ~~~\par ~~~\par ~~~
\tcblower
\tcbsubtitle[enlarge top initially by=-3.1mm]% -(middle+boxsep+tinydelta)
{My subtitle}
The lower part.
\end{tcolorbox}
\end{document}
This gives:
